Glycosylated il-7, preparation and uses

ABSTRACT

The present invention relates to new and improved interleukin-7 polypeptides, as well as compositions comprising the same, their preparation and uses. The invention more particularly relates to hyperglycosylated IL-7 polypeptides having improved properties, as well as their manufacture and therapeutic uses. The invention also discloses novel IL-7 polypeptides having modified amino acid sequences containing artificially created glycosylation site(s), as well as corresponding nucleic acid molecules, vectors and recombinant host cells. The invention also relates to the use of such polypeptides, cells or nucleic acids for curative or preventive treatment of mammalian subjects, including human subjects.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/996,176, filedJan. 18, 2008, which is the U.S. national stage application ofInternational Patent Application No. PCT/IB2006/002663, filed Jul. 19,2006, the disclosures of which are hereby incorporated by reference intheir entirety, including all figures, tables and amino acid or nucleicacid sequences.

The present invention relates to new and improved interleukin-7polypeptides, as well as to compositions comprising the same, theirpreparation and uses. The invention more particularly relates tohyperglycosylated IL-7 polypeptides having improved properties, as wellas their manufacture and therapeutic uses. The invention also disclosesnovel IL-7 polypeptides having modified amino acid sequences containingartificially created glycosylation site(s), as well as correspondingnucleic acid molecules, vectors and recombinant host or host cells. Theinvention also relates to the use of such polypeptides, cells or nucleicacids for curative or preventive treatment of mammalian subjects,including human subjects.

BACKGROUND OF THE INVENTION

B and T lymphocytes are the primary effector cells of the immuneresponses. Both cell classes are considered to derive ultimately fromhematopoietic stem cells in the mammalian bone marrow, via progenitor orprecursor cells representing distinguishable stages in thedifferentiation of each class. Mature T cells develop principally in thethymus, presumably from a precursor cell which migrates from the bonemarrow to the thymus at an early stage of T lymphocyte development.Lymphoid cells development is dependent on growth, survival anddifferentiation factors produced by various stromal cells. Numbers offactors are active on mature peripheral B and T cells, including IL-1,IL-2, IL-4, IL-5, interferon gamma, BSF-2, neuroleukin, transforminggrowth factor beta and IL-7.

“Interleukin-7” or “IL-7” refers to a mammalian endogenous secretoryglycoprotein which is capable of inducing proliferation of bonemarrow-derived lymphocyte progenitors and precursors, including thespecialized precursors known as pre-B cells. Originally derived from thestromal element of a bone marrow cell line, IL-7 is also secreted bythymic stromal cells, intestinal and other epithelial cells, somedendritic cells and follicular dendritic cells, keratinocytes andgenerally all lymphoid tissues. Alternative designations for thismolecule are “pre-B cell growth factor” and “lymphopoietin-1”.

EP0314415 (or U.S. Pat. No. 4,965,195) describes mammalian interleukin-7proteins and corresponding DNAs. Human IL-7 amino acid sequence containsthree putative N-linked glycosylation sites, located at Asn residues atpositions 70, 91 and 116. Transient recombinant expression of hIL-7(human IL-7) in COS cells allowed the visualization of r-huIL-7(recombinant human IL-7) as three protein bands of apparent molecularweight of about 20, 24 and 28 kDa (Cosman et al.; Lymphokine ReceptorInteractions; 1989; 179:229-236). Stable recombinant expression of hIL-7in BHK cells was also reported (Armitage et al.; The Journal ofImmunology; 1990; 144:938-941). However, the glycosylation status ofnaturally-occurring human IL-7, particularly the O-glycosylation status,has never been documented or studied, and the impact of theglycosylation profile on IL-7 properties has never been considered.Furthermore, unglycosylated mature human IL-7 (r-hIL-7) produced in E.coli, as described in EP0314415, exhibits a 17,387 Daltons molecularweight and displays a high activity in vitro on specific bioassays basedon the proliferation of various lymphocytes populations. Other cytokinesand growth factors, such as G-CSF, GM-CSF, IFN, HGF, etc, also displayfull therapeutic activity without glycosylation.

WO2004/018681 discloses an active conformer of human IL-7, comprisingthe following disulfide bridges: 1-4 (C2-C92), 2-5 (C34-C129) and 3-6(C47-C141), as well as methods of producing or characterizing the sameand the uses thereof.

IL-7 was originally disclosed as a cytokine whose principal activity wasthe induction of precursor B cell proliferation (Namen A. E. et al.;Journal of Experimental medicine; 1988; 167:988-1002). IL-7 has morerecently been disclosed as being involved in the survival andproliferation of thymocytes (T-Cells) during early stage of T-celldevelopment (Schluns K. S. et al.; Nature Immunology; 2000;1(5):426-432). IL-7 pathway is essential for lymphocyte developmentnotably on developing thymocytes (Maeurer M. J. et al.; Int. Rev.Immunol.; 1998; 16:309-22-Fry T. J. et al.; Blood; 2002; 99:3892-904).Fry and collaborators further identified IL-7 as a potent modulator ofthymic-independent T-cell regeneration in a multifactorial action (FryT. J. et al.; Blood; 2001; 97(6):1525-1533). IL-7 potently modulatesmature T cells and beside this effect on mature T cells, IL-7 mayinfluence the development of antigen presenting cells (Marquez C. etal.; J. Exp. Med.; 1995; 181:475-83). IL-7 is essential for memory Tcell regeneration, both in the CD4+ and CD8+ subsets (Kondrack R. M. etal.; J. Exp. Med.; 2003; 198:1797-806-Kaech S. M. et al.; Nat. Immunol.;2003; 4:1191-8).

IL-7 has thus a great therapeutic potential for use in the stimulationof the proliferation of T cell precursors, of antibody-secreting Bcells, in the stimulation of antigen driven T-cell peripheral expansion,and in the production of naïve T-cells as well as other hematopoieticcell types. A particularly interesting therapeutic use of active IL-7molecules is for immune reconstitution of lymphopenic patients: patientstreated for a cancer, patients having received a bone marrow or a stemcell transfer, patients presenting an acquired or genetic immunedeficiency, elderly patients or any patients having low CD4 count. Otherutilities reside in the ability of IL-7 to produce new naïve CD4 T-cellsor to expand specific pools in order to produce or increase specificimmune responses (vaccine enhancement).

In view of its therapeutic potentials, there is considerable interest indeveloping biologically active or improved IL-7 polypeptides that aresuitable for efficient therapeutic uses. In this respect, among thevarious cytokines and growth factors commercially available, some arepoorly immunogenic (e.g., Interferon alfa “IFNα”, granulocyte colonystimulating factor “G-CSF”) so that the corresponding drug substances donot require a very specific polypeptide purity other than conventionallevel usually accepted for recombinant proteins. In contrast, othergrowth factors are more immunogenic (e.g., Beta interferon “β-IFN”,Granulocyte Macrophage Colony stimulating factor “GM-CSF”) or theirspecific activity is so critical for life (e.g., Erythropoietin “EPO”)that drug substance polypeptide purity and profile must be specificallystudied and maintained inside narrow limits to preserve fromimmunogenicity.

IL-7 is a unique molecule. Due to its intrinsic immune-enhancingproperties, IL-7 used as a therapeutic agent is particularly prone totrigger anti-IL-7 immunogenicity (anti-IL-7 binding or neutralizingantibodies). This immunogenicity is deleterious for the long termtherapeutic activity of the protein. Anti-IL-7 antibodies can modifyIL-7 pharmacokinetic and neutralize its therapeutic activity.

Various IL-7 isoforms are involved in triggering anti-IL-7immunogenicity, among which: altered polypeptide sequences (e.g.,oxidized, reduced, deamidated or truncated forms), covalent or noncovalent IL-7 multimers, such as aggregated IL-7 molecules and the like.Therefore it is critical to define IL-7 polypeptides and drug substanceswhich are more stable, less prone to intermolecular aggregation, lessimmunogenic and still biologically active. Indeed, while the activity ofmost drugs is correlated with AUC parameter, the activity of IL-7 iscorrelated to half-life parameter and more particularly to meanresidence time.

SUMMARY OF THE INVENTION

The present invention discloses new and improved IL-7 polypeptides, drugsubstances and compositions. More particularly, the invention disclosesnovel IL-7 molecular species having a high degree of glycosylation andan oligosaccharide profile shifted to a higher molecular size withincreased sialylation and fucosylation of the carbohydrate moieties anda lower isoelectric point. The invention shows that these newoligosaccharide profiles confer improved chemical and pharmaceuticalstability to these new drug substances and a prolonged pharmacokineticprofile after in vivo administration, characterized by an increased meanresidence time (MRT), allowing a less frequent dosing schedule.

The present invention therefore provides novel highly glycosylated orhyperglycosylated IL-7 polypeptides having improved properties. Theinvention also discloses novel IL-7 polypeptides having modified aminoacid sequences containing artificially created glycosylation site(s), aswell as corresponding nucleic acid molecules, vectors and recombinanthost or host cells. The invention also relates to the use of suchpolypeptides, cells or nucleic acids for curative or preventivetreatment of mammalian subjects, including human subjects. The presentinvention thus discloses novel active IL-7 polypeptides, drug substancesand pharmaceutical compositions, which exhibit increased stability,reduced susceptibility to proteolysis and aggregation, advantageous invivo long term activity and reduced immunogenicity, thereby allowingimproved global or specific immune responses to be generated inmammalian subjects.

An object of this invention resides in a hyperglycosylated IL-7composition.

A further object of this invention resides in a purifiedhyperglycosylated IL-7 polypeptide.

Such hyperglycosylated IL-7 polypeptide contains at least threeN-glycosylated amino acid residues.

A further object of this invention relates to the use ofhyperglycosylated IL-7 for the manufacture of a medicament consisting ofsaid hyperglycosylated IL-7 and a pharmaceutically acceptable excipientor vehicle, for treating a mammalian subject.

A further object of this invention relates to the use of ahyperglycosylated IL-7 composition for the manufacture of a medicamentfor treating a mammalian subject.

A further object of this invention is a method of causing or stimulatingan immune response in a subject, comprising administering to the subjectan effective amount of a hyperglycosylated IL-7 composition.

A further object of this invention is a method for ex-vivo enhancingexpansion of T cells, which method comprises contacting T cells with ahyperglycosylated IL-7 polypeptide or composition, hereby enhancingexpansion of the T cells.

In a particular embodiment, the hyperglycosylated IL-7 composition is acomposition comprising at least 80%, preferably between 80% and 95%,IL-7 polypeptides which are glycosylated on at least three distinctamino acid residues. Such residues may be either naturally presentwithin an IL-7 polypeptide sequence and/or artificially createdglycosylation sites(s).

In a further particular embodiment, the hyperglycosylated IL-7composition is a composition comprising at least 80%, preferably between80% and 95%, IL-7 polypeptides which are glycosylated on from three upto eight distinct amino acid residues, including one O- and up to sevenN-glycosylation sites. Such residues may be either naturally presentwithin an IL-7 polypeptide sequence and/or artificially createdN-glycosylation sites(s).

In this regard, a further object of this invention relates to IL-7polypeptides having a modified amino acid sequence, wherein saidsequence comprises at least one artificially created glycosylation site.According to particular embodiments, the IL-7 polypeptides of thisinvention comprise 1, 2, 3 or 4 artificially created glycosylationsites, more preferably 1, 2 or 3; even more preferably 1 or 2. As willbe disclosed further, the artificially created glycosylation sites arepreferably N-linked glycosylation sites. The IL-7 polypeptides of thisinvention may be from any mammalian origin, particularly of humanorigin. Furthermore, such IL-7 polypeptides may comprise the sequence ofa mature IL-7 polypeptide, or further comprise additional amino acidresidues, such as a secretion peptide for instance. In addition, or inthe alternative, the IL-7 polypeptide is preferably a specific conformercomprising the following three disulfide bridges: Cys: 1-4 (Cys2-Cys92);2-5 (Cys34-Cys129); 3-6 (Cys47-Cys141). Specific examples of suchmodified IL-7 polypeptides comprise at least one amino acid modificationas disclosed in Table 1 below, or a combination thereof.

A further object of this invention resides in a nucleic acid moleculeencoding an IL-7 polypeptide as discussed above. The nucleic acidmolecule may be any DNA or RNA molecule, typically a cDNA molecule.

A further object of this invention resides in a nucleic acid moleculeencoding secretion signal comprising SEQ ID NO: 19.

A further object of this invention resides in a vector comprising anucleic acid molecule as defined above. The vector may be anyprokaryotic or eukaryotic vector, typically a eukaryotic vector, and maybe selected from a plasmid, episomal DNA, cosmid, viral vector,artificial chromosome, etc. The vector may comprise any regulatorysequence allowing proper expression of the coding nucleic acid in aselected host cell, e.g., a promoter, terminator, polyA, origin ofreplication, homologous region, intron, genes 5′ or 3′ untranslatedregions (UTR) etc.

The above nucleic acids and vectors may be used for instance to producerecombinant mammalian IL-7 polypeptides in various competent host cells,as well as for gene therapy purposes.

Another object of this invention resides in a recombinant host cellcomprising a nucleic acid or a vector as disclosed above. Such arecombinant cell may be prokaryotic or, more preferably, eukaryotic,such as a yeast, insect, plant or mammalian cell, for instance, morepreferably, recombinant host cell transduced to express or over expressa glycosyltransferase and/or a 2-6-sialyltransferase gene, e.g., fromhuman origin.

Another object of this invention resides in a drug substance comprisingan IL-7 polypeptide as described above, typically a hyperglycosylatedIL-7 polypeptide. More preferably, the drug substance contains less thanabout 10% of un- or mono-glycosylated IL-7 polypeptide and/or isessentially devoid of product-related impurities.

The invention also relates to the use of a drug substance as describedabove in the manufacture of a medicament (“drug product”) orpharmaceutical composition.

The invention further relates to a pharmaceutical composition comprisingan effective amount of an IL-7 polypeptide or composition or drugsubstance as described above and one or more pharmaceutically compatiblecarriers or excipients.

The invention also provides an antibody, as well as fragments orderivatives thereof, specifically immunoreactive with an IL-7polypeptide as defined above; hybridoma cell lines that produce saidantibody, as well as compositions suitable for diagnosis, assay ortherapy comprising said antibody, fragments or derivatives thereof.

A further aspect of this invention is a method of producing an IL-7polypeptide as described above, from prokaryotic or eukaryotic hostcells, as well as a method of detecting or measuring the presence ofsuch an IL-7 polypeptide in a sample, or to characterize a sample.

In a particular aspect, the method of producing an IL-7 polypeptide asdefined above comprises:

a) culturing a recombinant host cell as defined above, and

b) collecting the IL-7 polypeptide produced from said cell.

According to a preferred embodiment, expression is performed underconditions allowing efficient glycosylation motifs to be added to theIL-7 polypeptide, in particular sialic acid residues.

In a further preferred embodiment, the production is performed in afed-batch or perfusion mode maintaining the cells at the end ofexponential growth phase. Such conditions increase the quality ofpost-translational modifications and contribute to a higher degree ofsialylation per IL-7 polypeptides. According to particular embodiments,the encoding nucleic acid comprises a secretion signal and/or anoptimized nucleic acid sequence and/or the host cell is a eukaryotichost cell (e.g., a mammalian or insects or yeast cell).

Another object of the invention relates to the use of an IL-7polypeptide or a hyperglycosylated IL-7 composition, as defined above orobtained by a method as described above, for the manufacture of apharmaceutical composition to cause or modulate an immune response in asubject, particularly to induce prolonged lymphopoiesis stimulationand/or to amplify an immune response.

The invention also relates to the use of an IL-7 polypeptide as definedabove or obtained by a method as described above, for the manufacture ofa pharmaceutical composition to prevent or treat a disease associatedwith an immunodeficiency.

As will be discussed below, the polypeptides of this invention exhibitan extended plasma half-life and mean residence time, which favor invivo receptor interaction and activity, and/or an improved stabilityand/or a lesser long term immunogenicity, thereby allowing their uses totreat a variety of pathological conditions in mammalian subjects,particularly in human subjects.

LEGENDS TO THE FIGURES

FIG. 1: Plasmid ph-pgk.EP7-hIL-7:

ef1a pA: “elongation factor 1 alpha” poly A sequence; hgh pA: “humangrowth hormone” poly A sequence; SpA: synthetic polyA sequence; hph:hygromycin resistance; Amp: Ampicillin resistance; MAR rabbit βglobin:putative rabbit βglobin “Matrix Attachment Region”; pr. tk: thymidinekinase promoter, sv40 enh.: sv40 enhancer; pr pgk: phosphoglyceratekinase promoter; 5′UTRint1: 5′ untranslated region comprising a chimericintron (hBglobin-immunoglobuline); EP7-hIL7: optimized human IL-7 cDNAupstream from EP7 signal peptide.

FIG. 2: Plasmid pBh-pgk.EP7-hIL-7: Bc12: Bc12 cDNA; IRES: InternalRibosome Entry Site

FIG. 3: Expression of recombinant hIL-7 in mammalian cells cultured inBioreactor from day 1 (D1) to day 10 (D10) Western blot of intracellularversus secreted IL-7.

FIG. 4: Chromatographic fractionation of rec-hIL-7glycoforms throughoutpurification process: SDS PAGE analysis of the protein content in thedifferent elution fractions (B1-B10). IL-7 glycoforms were separatedduring both the capture and HIC steps. Buffer gradients were used so asto elute differentially the hIL-7 glycoforms according to their slightlydifferent physico-chemical properties. Fractionation and subsequentselection of adequate fraction allowed an enrichment of the fully threeglycosylated recombinant hIL-7 (3 N- or 2 N-associated with 1O-sugarmoiety). MWM, protein molecular weight markers (10; 15; 20; 25; 37; 50;75; 100; 150; 250 kDa); B1-B10, elution fractions; B1-B4, elutionfractions retained for further purification; CT, fraction B1 obtainedfrom a culture of HEK293 cell line transfected with the same optimizedhIL-7 cDNA.

FIG. 5: Analyses of the purified recombinant hIL-7 on SDS PAGE. Samplesof the purified recombinant hIL-7 were loaded on SDS PAGE under reducingconditions. Gels were revealed by:

A. Coomassie staining

B. Western blot

MWM: molecular weight markers (10; 15; 20; 25; 37; 50; 75; 100; 150; 250kDa). Lane 1: HG-37-147; Lane 2: HG-40-104; Lane 3: HG-hIL-7; Lane 4: E.coli hIL-7.

FIG. 6: Comparative SDS-PAGE apparent molecular weight of purifiedglycosylated rec-hIL-7 products.

Lane M=Molecular weight marker, Lane 1=schematic representation ofpurified product as described by Namen et al. in U.S. Pat. No. 5,328,988(about 25 KDa), Lane 2=CHO rec-sIL-7 product purified by the Applicant,Lane 3=CHO rec-hIL-7 product purified by the Applicant, Lane 4=hIL-7 1N-and 2N-glycoforms as standard for apparent molecular weight comparison,Lane 5=E. coli rec-hIL-7 product purified by the Applicant (CYT 99 007).

FIG. 7: Analyses of the purified recombinant human IL-7 on SDS PAGEunder reducing conditions after deglycosylation.

Samples of the purified recombinant glycosylated human IL-7 weredigested by PNGase F: Kinetic samples from 2 minutes to 24 h were loadedon gel.

Another sample (OO/N) was digested over 24 h with PNGaseF+O-glycosidase/f3(1-4)galactosidase/neuraminidase/N-Acetylglucosaminidase.

As a control, glycosylated human IL-7 and E. coli human IL7 were alsoloaded on the gel.

3N+1O, 2N+1O, 1N+1O, 1O glycoforms and deglycosylated human IL7 werethen separated according to their MW estimated on the gel at 33, 27, 24,18 and 17 kDa, respectively.

FIG. 8: Mass spectrum analysis of different purified rec-hIL-7Glycoforms.

-   -   23 kDa (23179 Da): Rec-hIL-7(CHO S,2N+3N)    -   25 kDa (25127 Da): Rec-hIL-7(CHO S,3N)

FIG. 9: 2D electrophoresis analysis of the purified recombinanthyperglycosylated human IL-7 polypeptide. After Iso Electric Focusing(pH range 3-10), glycoforms were separated on SDS PAGE under reducingconditions (Coomassie blue staining)

FIG. 10: Mass spectrum analysis of recombinant hIL-7 N-glycancomplexity. Purified glycosylated hIL-7 samples were enzymaticallydigested with an endoglycosidase such as peptide-N-glycosidase F(PNGaseF, Roche). Released N-linked oligosaccharides, released proteinsample by enzymatic digestion were separated from the peptide structureand analysed by MALDI-TOF Mass Spectrometry. The m/z valuescorresponding to each peak were searched against international databasesand allowed accurate identification of the panel of N-Glycans on thehIL-7 molecule.

FIG. 11: Characterization of O-glycans on recombinant hIL-7, usingspecific lectins (lectin blot). After separation of protein samples andblotting to membrane, products were revealed with either one of the twolectins (MAA, from Maackia amurensis, PNA, from Arachis hypogea). Lane1: standard protein fetuin, a sialilated protein, Lane 2, fetuin treatedwith a sialidase, Lane 3, hIL-7, Lane 4, hIL-7 treated with sialidase.

FIG. 12: Lectin affinity of hyperglycosylated IL-7 polypeptide (ELISAscreen). Lectins (LEA from Lycopersicon esculentum, WGA from Triticumvulgare, UEA.I from Ulex europeus, MAA from Maackia amurensis, ACA fromAmaranthus caudatus, AIA from Artocarpus intergrifolia, ABA fromAgaricus bisporus, PHA.L from Phaseolus vulgaris having)—cated platesused to bind identical amounts of recombinant purified hIL-7preparations. Amounts of bound IL-7, depending on the lectin specificityto the glycan moieties, were revealed by a specific anti hIL-7 antibody(Ab) coupled to Biotin. The Lectin-IL-7-Ab sandwich was revealed with astreptavidin-peroxidase conjugate.

FIG. 13: Bioassay of recombinant human IL-7 activity. Dose-responsekinetics of PB-1 cell growth induced by unglycosylated r-hIL-7(expressed in E. coli) or highly glycosylated r-hIL-7 (produced inmammalian cells).

FIG. 14: Bioassay of recombinant human IL-7 activity. Dose-responsekinetic data and curves obtained routinely in a typical bioassay: thePB-1 cell growth was induced by unglycosylated r-hIL-7 (expressed in E.coli), highly glycosylated or hyperglycosylated r-hIL-7 (produced inmammalian cells). (Data points represent the mean±SD of triplicatedetermination).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to hyperglycosylated IL-7 compositions,their manufacture and their uses in the pharmaceutical industry. Thepresent invention, for the first time, shows that the activity and/orproperties of IL-7 may be enhanced depending on the glycosylationprofile of the polypeptide. The present invention also discloses,surprisingly and contrary to in vitro data, that the best in vivoactivity can be achieved with IL-7 polypeptides having at least 2 topreferably 3 occupied N-linked glycosylation sites and one O-linkedglycosylation site and maximized terminal sialylation of theoligosaccharide moieties. The present invention also disclosesartificially created hyperglycosylated IL-7 polypeptides, which exhibitprolonged activity (thereby allowing a reduced administrationfrequency), and/or a decreased long-term immunogenicity. Considering theutility of IL-7, such polypeptides and compositions represent highlyvaluable and useful active molecules for use in regulating an immuneresponse in a subject, including a human subject.

A first object of this invention thus resides in a hyperglycosylatedIL-7 composition.

A further object of this invention relates to the use ofhyperglycosylated IL-7 for the manufacture of a medicament consisting ofsaid hyperglycosylated IL-7 and at least one pharmaceutically acceptablecarrier or excipient, for treating a mammalian subject.

A further object of this invention is a method for causing orstimulating an immune response in a subject, comprising administering tothe subject an effective amount of a hyperglycosylated IL-7 composition.

A further object of this invention relates to the use of ahyperglycosylated (and preferably highly sialylated) IL-7 compositionfor the manufacture of a medicament for causing or stimulating an immuneresponse in a subject.

IL-7 Polypeptide

Within the context of the present invention, an “IL-7 polypeptide”designates a mammalian (e.g., human, simian, bovine, equine, feline orcanine) IL-7 polypeptide. More preferably, the IL-7 polypeptide is ahuman IL-7 polypeptide, especially for uses as a therapeutic or vaccine.Alternatively, especially for use in non human primate experiments or inveterinary applications, the IL-7 polypeptide may be any other mammalianIL-7 polypeptides such as a simian IL-7 polypeptide or a caninepolypeptide.

Preferred human IL-7 polypeptides of this invention comprise an aminoacid sequence as described in EP 314 415 or in WO2004/018681 A2, as wellas any natural variants and homologs thereof. The sequence of human IL-7is also available on gene banks. The typical wild-type protein comprises152 amino acids and, optionally, an additional N-terminal methionineresidue (SEQ ID NO: 1). Variants thereof include, more preferably,natural allelic variants resulting from natural polymorphism, includingSNPs, splicing variants, etc. In a specific embodiment, the term IL-7polypeptide is meant to designate a polypeptide having the sequence ofSEQ ID NO: 1 or natural variants thereof.

In a further embodiment, the IL-7 polypeptide is a canine IL-7polypeptide. In this regard, the invention discloses, for the firsttime, the sequence of an isolated IL-7 polypeptide, which represents afurther object of this application. In particular, the invention relatesto an IL-7 polypeptide comprising amino acid sequence depicted in SEQ IDNO: 7, as well as any natural variants, homologs or distinctivefragments thereof. The term “variants” or “homologs” refer topolypeptides that differ from SEQ ID NO:7 by a deletion, substitution oraddition of one or a limited number of amino acids. Preferably suchvariants or homologs show a percentage of identity that is superior to85%, preferably superior to 90%, preferably superior to 95%, mostpreferably superior to 98% with SEQ ID NO:7.

The IL-7 polypeptide used in the present invention is preferably arecombinant IL-7. The term “recombinant”, as used herein, means that thepolypeptide is obtained or derived from a recombinant expression system,i.e., from a culture of host cells (e.g., microbial or insect or plantor mammalian) or from transgenic plants or animals engineered to containa nucleic acid molecule encoding an IL-7 polypeptide. “Microbial” refersto recombinant proteins made in bacterial expression systems.“Mammalian” refers to recombinant glycoproteins made in mammalianexpression systems. As will be discussed below, all of these host cellsshould preferably express either naturally or after transgenesis anappropriate glycosyltransferase and/or sialyltransferase gene. IL-7polypeptide can also be glycosylated through the use of appropriate invitro or in vivo glycosyltransferase and/or sialyltransferase molecules,or by grafting oligosaccharide structures.

A specific example of a human IL-7 polypeptide is a polypeptide of SEQID NO: 1 comprising the disulfide bridges Cys2-Cys92; Cys34-Cys129 andCys47-Cys141.

Also, IL-7 polypeptides of the present invention may comprise thesequence of a mature IL-7 polypeptide, or further comprise additionalamino acid residues, such as a secretion peptide for instance. Preferredexamples of such secretion peptides include, without limitation, asignal peptide selected from the group consisting of the EPO signalpeptide, SEAP signal peptide, IgGkappa signal peptide,Lactotransferin/vitronectin signal peptide, VIP/vitronectin signalpeptide and cytostatin bis signal peptide. The sequence of these signalpeptides is set forth, respectively, in SEQ ID NO 13 to 18. In aspecific embodiment, the signal peptide is a hybrid construct made bythe inventors, between sequences derived from the EPO and IL-7 signalpeptides. The sequence of this signal peptide, termed EPy7 or EP7, isset forth in SEQ ID NO: 19 and represents a particular object of thisinvention.

Hyperglycosylated IL-7 and Compositions

Within the context of the present invention, the term “hyperglycosylatedIL-7” designates an IL-7 polypeptide having at least three occupiedglycosylation sites, i.e., having at least three glycosylated amino acidresidues.

A “glycosylation site” designates any amino acid residue or region in apolypeptide which is subject to glycosylation, i.e., the attachment of acarbohydrate structure. Such sites are typically N-glycosylation sites(i.e., any amino acid residue or region in a polypeptide which allowsthe attachment of a carbohydrate structure through N-linkage) and/orβ-glycosylation sites (i.e., any amino acid residue or region in apolypeptide which allows the attachment of a carbohydrate structurethrough O-linkage). Consensus sequences for glycosylation sites areknown per se in the art. As an illustration, a consensus N-glycosylationsite typically has the following structure: Asn-X-Ser/Thr, where X isany amino acid except Proline. As will be disclosed below, suchglycosylation sites may be either naturally present within an IL-7polypeptide sequence and/or artificially added or created within saidsequence.

The term “hyperglycosylated IL-7 composition” designates an IL-7composition in which at least 80% of IL-7 polypeptides have at leastthree occupied glycosylation sites, i.e., having at least threeglycosylated amino acid residues. Preferably, such a compositioncomprises at least 80% of IL-7 polypeptides that are glycosylated, atleast, at three N-glycosylation sites(s) and, optionally, at oneO-glycosylation site. Such a composition is most preferably essentiallydevoid of un- or mono-glycosylated IL-7 polypeptides, and thus comprisesat most 20% of bi-glycosylated IL-7 polypeptides. In a preferredembodiment, a hyperglycosylated IL-7 composition designates an IL-7composition in which at least 90% of IL-7 polypeptides are glycosylatedat three N-glycosylation sites(s) and, optionally, at oneO-glycosylation site. Such a composition is most preferably essentiallydevoid of un- or mono-glycosylated IL-7 polypeptides, and thus comprisesat most 10% of bi-N-glycosylated with or without mono-β-glycosylatedIL-7 polypeptides.

IL-7 primary amino acid sequence comprises three putativeN-glycosylation sites, namely Asparagine (Asn) residues at positions 70,91 and 116 (with respect to the human wild type sequence, see SEQ ID NO:1). Furthermore, the present invention shows that the IL-7 sequence alsocontains one O-glycosylation site, namely Threonine (Thr) residue atposition 110. In a particular embodiment, a hyperglycosylated IL-7polypeptide of the present invention is an IL-7 polypeptide having theabove three N-glycosylation sites occupied associated or not to oneoccupied O-glycosylation site and a hyperglycosylated IL-7 compositionis an IL-7 composition in which at least 80% of IL-7 polypeptides areglycosylated at the above N-glycosylation sites and, optionally, also atthe O-glycosylation site.

In a particular embodiment, a hyperglycosylated IL-7 polypeptide maycomprise additional artificially added or created glycosylation sites.Accordingly, a hyperglycosylated IL-7 polypeptide of the presentinvention is an IL-7 polypeptide having at least three N-glycosylationsites and one O-glycosylation site occupied, said sites being eithernaturally-occurring and/or artificially added/created; and ahyperglycosylated IL-7 composition is an IL-7 composition in which atleast 80% of IL-7 polypeptides are glycosylated at four glycosylationsites(s) at least, said sites being either naturally-occurring and/orartificially added/created.

In this regard, the present invention now discloses IL-7 polypeptideshaving a modified amino acid sequence, wherein said sequence comprisesat least one artificially created glycosylation site. According toparticular embodiments, the IL-7 polypeptides of this invention comprise1, 2, 3 or 4 artificially created glycosylation sites, more preferably1, 2 or 3; even more preferably 1 or 2.

The artificially created glycosylation sites are preferably N-linkedglycosylation sites. Consensus N-glycosylation sites typically have thefollowing structure: Asn-X-Ser/Thr, where X is any amino acid exceptProline.

The glycosylation sites may be created or added chemically fromassembled synthetic oligonucleotides or using several techniquesincluding mutagenesis methods at various positions within IL-7 primaryamino acid sequence, and following techniques known per se in the art.Because the modified IL-7 polypeptide shall retain the ability to bindan IL-7 receptor, the glycosylation site(s) is (are) most preferablycreated within region(s) or domain(s) of the IL-7 polypeptide sequencewhich do(es) not alter the ability of IL-7 to bind an IL-7 receptor.More preferably, the site(s) is (are) introduced outside of the alphahelices of the polypeptide, preferably except at immediate proximity ofthe glycine residues. Preferably, they are introduced in the mostflexible region, avoiding regions that are more rigid and important fortertiary structure of the polypeptide. Preferably, the creation of aglycosylation site does not affect any Cystein residue involved in adisulfide bridge (e.g., Cys 2, 34, 47, 92, 129 and 141), nor anycritical residue involved in the interaction of IL-7 polypeptide withits cognate receptor (e.g., Ser 19, Leu 23 and 77, Tyr 12, Val 15, Gln22, Lys 81 and Glu 84), nor any conserved residue involved in theactivity of the polypeptide (e.g., Arg 133, Gln 136, Glu 137, Lys 139and 144, Thr 140 and Asn 143). The glycosylation sites are typicallycreated by mutation, deletion or addition of one or several amino acidresidues within the primary sequence of a reference IL-7 polypeptide, tocreate a typical consensus glycosylation site.

In a particular embodiment, the present invention relates to IL-7polypeptides comprising the sequence of a human (or mammalian) IL-7polypeptide comprising one or several amino acid modifications selectedfromLys28Asn-Ile30Ser-Ile30Thr-Ile30Asn-Ser32Thr-Leu35Ser-Leu35Thr-Glu38Ser-Glu38Thr-Phe39Ser-Phe39Thr-Phe42Ser-Phe42Thr-Glu52Ser-Glu52Thr-Val82Asn-Glu84Thr-Glu84Ser-Lys97Asn-Arg99Thr-Arg99Ser-Ala102Asn-Leu104Thr-Leu104Ser-Leu104Asn-Glu106Thr-Glu106Ser-Leu128Ser-Leu128Thr-Ile145Asn-Met147Thr-Met147Ser-Met147Asn-Thr149Ser(or from corresponding positions in other mammalian IL-7 polypeptides).

Specific examples of (human) IL-7 polypeptides of this inventioncomprise the amino acid modifications disclosed in Table 1 below:

TABLE 1 IL-7 polypeptide analog amino acid changes HG28a Lys28Asn;Ile30Ser HG28b Lys28Asn; Ile30Thr HG30 Ile30Asn; Ser32Thr HG33a Leu35SerHG33b Leu35Thr HG36a Glu38Ser HG36b Glu38Thr HG37a Phe39Ser HG37bPhe39Thr HG40a Phe42Ser HG40b Phe42Thr HG50a Glu52Ser HG50b Glu52ThrHG82a Val82Asn; Glu84Ser HG82b Val82Asn; Glu84Thr HG97a Lys97Asn;Arg99Ser HG97b Lys97Asn; Arg99Thr HG102a Ala102Asn; Leu104Ser HG102bAla102Asn; Leu104Thr HG104a Leu104Asn; Glu106Ser HG104b Leu104Asn;Glu106Thr HG126a Leu128Ser HG126b Leu128Thr HG145a Ile145Asn; Met147SerHG145b Ile145Asn; Met147Thr HG147 Met147Asn; Thr149Ser

The above amino acid modifications create N-glycosylation sites withoutsubstantially altering the binding affinity of IL-7, thereby creatingimproved IL-7 polypeptides according to the present invention.

The term “without substantially altering the binding affinity” meansthat the binding affinity is not altered or may be reduced withoutimpacting the in vivo effects resulting from the interaction with thereceptor. When quantified in vitro, the binding affinity may be reducedby less than 50%, preferably less than 40%, preferably less than 30%,preferably less than 20%, preferably less than 5%.

Also, the above modifications can be combined to create severaladditional glycosylation sites within an IL-7 polypeptide of thisinvention. In this regard, the invention relates to any biologicallyactive IL-7 polypeptide having the primary sequence of (human ormammalian) interleukin-7 modified by the addition of at least from oneto four additional (N-linked) glycosylation sites. A further preferredembodiment of this invention is a biologically active IL-7 polypeptidecomprising the primary sequence of interleukin-7 comprising one or twoadditional (N-linked) glycosylation sites.

Most preferred IL-7 polypeptides of this invention are biologicallyactive, i.e., they are capable of binding to an interleukin-7 receptorand show in vitro activity in a specific bioassay and/or have anincreased mean residence time (MRT) in vivo.

Comparison of activities between the non glycosylated standard and thehyperglycosylated IL-7 was addressed in a dose/response bioassay studyin which an ED50 value corresponds to a dose equal to one half ofmaximal activity.

Hyperglycosylation, usually leads to decreased activity in the bioassay,which does not translate into decreased in vivo activity. In the presentsituation, the extended kinetic profile will in fact improve the in vivoactivity.

In spite of the fact that ED50 does not reflect activity of the IL-7 invivo, it allows comparison between different samples with the same levelof glycosylation. In this frame, typical ED50 values for a nonglycosylated standard were ranging between 0.5 to 2.0 ng IL-7/ml whilehyperglycosylated ED50 values were ranging between 1.5 to 3.5 nghyperglycosylated IL-7/ml.

The hyperglycosylated IL-7 polypeptides of the invention show animproved stability, and an in vivo extended half-life and mean residencetime in mammalian hosts. The term “improved stability”, “extendedhalf-life and mean residence time” is to be understood in comparison tonon-glycosylated forms. Preferably the increase of half-life is at leastabout 3×, preferably at least about 5 to 20×. Mean residence time (MRT)means the average of the residence time of each IL-7 molecule in theblood of patients after initial dosing. Preferably, the increase of MRTis at least about 2×, or preferably at least about 4 to 10× compared tothe MRT of non-glycosylated forms.

For instance the plasmatic half-life of the hyperglycosylated form wasshown to be in the range of 30 to 40 hours, whereas the plasmatichalf-life of the non-glycosylated form is usually 5 to 8 hours (whenboth forms are administered in the same conditions, i.e. in oneinjection, subcutaneously).

The mean residence time (MRT) was around 40 hours versus around 10 hourswith the non glycosylated form.

It should be understood that the invention also encompasses anydistinctive fragment of an IL-7 polypeptide of this invention, i.e., anyfragment comprising an amino acid modification as disclosed above, orany fragment comprising an artificially created glycosylation site asdisclosed above. Such fragments typically contain at least 5 amino acidresidues, typically at least 8, 9, 10, 11, 12 or 15 residues, and maycontain up to 20, 30, 40, 50 or more consecutive amino acid residues.Such fragments may be used as antagonists or as immunogens, to generatespecific antibodies.

Also, while the above amino acid positions have been given by referenceto the human IL-7 polypeptide sequence, it should be understood that thepresent invention also encompasses IL-7 polypeptides having the primarysequence of mammalian IL-7 modified by homologous mutations in mammaliansequences based on sequence alignment against human sequence.

A preferred embodiment of this invention relates to new biologicallyactive IL-7 polypeptides comprising one or several amino acidmodification(s) selected from the group consisting of:

Phe39Ser-Phe39Thr-Phe42Ser-Phe42Thr-Leu104Asn-Glu106Thr-Glu106Ser-Leu128Ser-Leu128Thr-Met147Asnand a combination thereof, or a distinctive fragment thereof.

Most preferred modified IL-7 polypeptides of this invention aredisclosed in Table 2 below:

TABLE 2 IL-7 polypeptide analog amino acid changes HG37a Phe39Ser HG37bPhe39Thr HG40a Phe42Ser HG40b Phe42Thr HG104a Leu104Asn; Glu106SerHG104b Leu104Asn; Glu106Thr HG126a Leu128Ser HG126b Leu128Thr HG147Met147Asn; Thr149Ser

A further particular object of this invention is a hyperglycosylatedIL-7 polypeptide comprising a primary amino acid sequence as disclosedabove.

Oligosaccharide Units

The structure and number of oligosaccharide units attached to aparticular glycosylation site in a hyperglycosylated IL-7 polypeptide ofthis invention can be variable. These may be, for instance, N-acetylglucosamine, N-acetyl galactosamine, mannose, galactose, glucose,fucose, xylose, glucuronic acid, iduronic acid and/or sialic acids.

More preferably, hyperglycosylated IL-7 polypeptides comprise (or areenriched in) N-linked and/or O-linked carbohydrate chain(s) selectedfrom:

-   -   a) a mammalian type sugar chain, preferably of the type        expressed by CHO cells;    -   b) a sugar chain comprising a complex N-carbohydrate chain        (e.g., a triantenary or biantenary structure), more preferably        containing high mannose and acetylglucosamine molecules and high        terminal sialic acid residues;    -   c) a sugar chain comprising an O-carbohydrate chain without and        preferably with a terminal sialic acid residue;    -   d) a sugar chain sialylated by alpha-2,6-sialyltransferase or        alpha-2,3-sialyltransferase; and/or    -   e) a sialylated sugar chain displaying between 3 to 30        sialyl-N-acetylgalactosamine, preferably 7 to 23.

Particularly preferred carbohydrate chain(s) comprise a triantenary orbiantenary structure with partial or complete terminal sialylation.Further preferred carbohydrate chains comprise triantenary structuresand tri or bi-sialylation, and/or a diantenary structure withdisialylation. Examples of such carbohydrates are disclosed in Table 4,including motifs #2420, 2623, 2785 and 3092.

According to a further specific embodiment, the hyperglycosylatedinterleukin-7 polypeptide of this invention has an average isoelectricpoint inferior to 6.5 and an average apparent molecular weight superiorto 27 kDa, between 28 KDa and 65 KDa (theroretical for a 7N+1Oglycosylation), preferably between 28 KDa and 35 KDa (as shown for a3N+1O glycosylation), by gel electrophoresis (confirmed by Western blot)which is translated to 25 kDa by mass spectrometry analysis.

In a particular embodiment, the hyperglycosylated IL-7 polypeptide ofthis invention is produced by a mammalian glycosylation mutant thatstably expresses α2,6 sialyltransferase and presents a deficiency inCMP-Neu5Ac Hydrolase activity, preferably a CHO glycosylation mutant.Such glycosylation typically includes N-acetyl glucosamine, N-acetylgalactosamine, mannose, galactose, glucose, fucose, xylose, glucuronicacid, iduronic acid and/or sialic acids.

In an other embodiment, the hyperglycosylated IL-7 polypeptide isproduced by recombinant technology in a human host cell, which may beselected from human stromal or epithelial cell lines, HEK-293 (HumanEmbryonic Kidney), HER (Human Embryonic Retina), HEK (Human EpidermalKeratinocytes), human thymus or human cortical epithelial cell lines,human bone marrow or human bone marrow stromal cell lines.

Most preferred hyperglycosylated interleukin-7 polypeptides of thisinvention display the following feature(s):

a) they have improved secretion profile and production rate inrecombined productive cell lines; and/or

b) they contain a high degree of sialic acid residue per IL-7polypeptide, leading to decrease isoelectric point value and to improvemean residence time; and/or

c) they are protected from inter-molecular aggregation; and/or

d) they have reduced susceptibility to proteolysis; and/or

e) they contain masked antigenic sites, reflecting reduced immunogenicliability, reduced vulnerability to APC (antigen presenting cells)capture, processing and presentation through a MHCII molecule; and/or

f) they have increased chemical stability; and/or

g) they have an extended biological half-life in vivo (Long actingisoform of IL-7) compared to the unglycosylated parent peptide; and/or

h) they have an increased in vivo pharmacological activity compared tounglycosylated parent protein, mostly due to a better mean residencetime (MRT); and/or

i) they permit less frequent dosing schedule, from three/four times aweek down to two or once a week or once every fortnight for the morelong acting products; and/or

j) they display an improved pharmacokinetic profile (decreased peakconcentration and improved mean residence time); and/or

k) they display an average molecular weight above 25 KDa as determinedfrom Mass Spectrometry analysis or 27 KDa as determined from SDS-PAGEanalysis and an average isoelectric point below 6.5.

The polypeptides of this invention may be in the form of a monomer, orassociated or complexed with a particular compound of choice. In thisregard, in a particular embodiment, the IL-7 conformer is associated tothe hepatocyte growth factor (“HGF”), as a heterodimer. The heterodimermay be obtained chemically, by complexation or by recombinant technology(i.e., by genetic fusion).

In an other particular embodiment, the IL-7 polypeptide is functionallyattached to a Fc portion of an IgG heavy chain, typically through apeptide hinge region. Such fusion molecules have potentially increasedstability and half-life in vivo. The IgG moiety is most preferably ahuman IgG1 or IgG4.

In an other particular embodiment, the IL-7 polypeptide is functionallyassociated to a human serum albumin (“HSA”) or a portion of a HSA, as afusion protein. Such fusion molecules have potentially increasedstability and prolonged half-life in vivo.

A further object of this invention is a hyperglycosylated IL-7composition. Such compositions preferably comprise at least 80%,preferably between 80% and 95%, IL-7 polypeptides which are glycosylatedon at least three distinct amino acid residues, which may be naturallypresent within an IL-7 polypeptide sequence (e.g. consensus N-linked andO-linked carbohydrate sites) and/or artificially created glycosylationsites(s), as discussed above.

According to particular, specific, embodiments, the invention relates tohyperglycosylated IL-7 compositions comprising:

-   -   a) a majority (>80%, preferably more than 90%, most preferably        more than about 95%) of interleukin-7 glycosylated on the 3        consensus N-linked carbohydrate sites (Asn 70/91/116) and        further glycosylated or not on 1 O-linked carbohydrate site (Thr        110); preferably, the composition contains a minority (<20%,        preferably less than about 10%) of interleukin-7 glycosylated on        2 consensus N-linked carbohydrate sites only (associated or not        to 1 O-linked carbohydrate site) and/or is essentially devoid of        mono- or unglycosylated protein; or    -   b) a majority (>80%, preferably more than 90%, most preferably        more than about 95%) of a biologically active interleukin-7        analog, having the IL-7 primary amino acid sequence modified to        introduce one additional site of glycosylation, glycosylated on        4 N-linked carbohydrate sites and further glycosylated or not on        1 O-linked carbohydrate site (Thr 110); preferably the        composition contains a minority of the same analog (<20%,        preferably less than about 10%) glycosylated on 3 or 2 N-linked        carbohydrate sites only (associated or not to 1 O-linked        carbohydrate site) and/or is essentially devoid of mono- or        unglycosylated protein; or    -   c) a majority (>80%, preferably more than 90%, most preferably        more than about 95%) of an interleukin-7 biologically active        analog, having the IL-7 primary amino acid sequence modified to        introduce two additional sites of glycosylation, glycosylated on        5 N-linked carbohydrate sites and further glycosylated or not on        1 O-linked carbohydrate site (Thr 110); preferably the        composition contains a minority of the same analog (<20%,        preferably less than about 10%) glycosylated on 4, 3 or 2        N-linked carbohydrate sites only associated or not to 1 O-linked        carbohydrate site and/or is essentially devoid of mono- or        unglycosylated protein; or    -   d) a majority (>80%, preferably more than 90%, most preferably        more than about 95%) of an interleukin-7 biologically active        analog, having the IL-7 primary amino acid sequence modified to        introduce three additional sites of glycosylation, glycosylated        on 6 N-linked carbohydrate sites and further glycosylated or not        on 1 O-linked carbohydrate site (Thr 110); preferably the        composition contains a minority of the same analog (<20%,        preferably less than about 10%) glycosylated on 5, 4, 3 or 2        N-linked carbohydrate sites only associated or not to 1 O-linked        carbohydrate site and/or is essentially devoid of mono- or        unglycosylated protein; or    -   e) a majority (>80%, preferably more than 90%, most preferably        more than about 95%) of an interleukin-7 biologically active        analog, having the IL-7 primary amino acid sequence modified to        introduce four additional sites of glycosylation, glycosylated        on 7 N-linked carbohydrate sites and further glycosylated or not        on 1 O-linked carbohydrate site (Thr 110); preferably the        composition contains a minority of the same analog (<20%,        preferably less than about 10%) glycosylated on 6, 5, 4, 3 or 2        N-linked carbohydrate sites only associated or not to 1 O-linked        carbohydrate site and/or is essentially devoid of mono- or        unglycosylated protein.

The invention also relates to pharmaceutical compositions comprising theabove compositions as the active substance.

Nucleic Acids

A further object of this invention resides in a nucleic acid moleculeencoding an IL-7 polypeptide as discussed above. The nucleic acidmolecule may be any DNA or RNA molecule, typically a cDNA molecule.

A specific object of this invention is a nucleic acid comprisingnucleotide residues 79 to END of SEQ ID NO: 2, as well as distinctivefragments and the complementary strand thereof.

A further object of this invention is a nucleic acid comprising SEQ IDNO: 4, as well as any distinctive fragment thereof, variants thereof(having at least 90% identity with SEQ ID NO: 4), and the complementarystrand thereof.

A further object of this invention is a nucleic acid comprising SEQ IDNO: 6, as well as any distinctive fragment thereof, variants thereof(having at least 90% identity with SEQ ID NO: 6) and the complementarystrand thereof A specific object of this invention is a nucleic acidcomprising nucleotide residues 79 to END of SEQ ID NO: 6, as well asvariants thereof (having at least 90% identity with SEQ ID NO: 6) andthe complementary strand thereof. The invention also encompasses apolypeptide encoded by such sequences (e.g., SEQ ID NO: 7).

The term “variant” as used above in relation to a nucleic acid morespecifically designates a nucleotide sequence that hydridizes to thereference sequence under stringent condition and/or encodes apolypeptide having the same type of activity as the polypeptide encodedby the reference sequence. Most preferred variants exhibit at leastbetween 92 and 99% (e.g., 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%)identity with the reference sequence.

A further specific object of this invention is a nucleic acid comprisingthe sequence of:

CTG AAT AAC GAA ACT AAC SEQ ID NO: 8 AAC TTC ACT AAG SEQ ID NO: 9 GCCAAC GGT ACC SEQ ID NO: 10 CTG AAC GAC AGC TGT, SEQ ID NO: 11 or ATC TTGAAC GGG, SEQ ID NO: 12or a combination thereof.

Specific examples of nucleic acids of this invention comprise thenucleotide sequence as set forth in any one of SEQ ID NOs: 8 to 12.

A further object of this invention resides in a cloning and/orexpression vector comprising a nucleic acid molecule as defined above.The vector may be any prokaryotic or eukaryotic vector, typically aneukaryotic vector, and may be selected from a plasmid, cosmid, viralvector, artificial chromosome, etc. The vector may comprise anyregulatory sequence allowing proper expression of the coding nucleicacid in a selected host cell, e.g., a promoter, terminator, polyA,origin of replication, integration region (e.g., homologous region),intron, UTR sequences, marker gene, etc.

A particular object of this invention is an expression vector comprisinga nucleic acid molecule as defined above, including a signal peptide,operably linked to regulatory elements allowing expression of saidnucleic acid in a mammalian host or host cell.

Preferred regulatory elements include a promoter, which may be selected,without limitation, from viral, cellular and synthetic promoters,including constitutive, tissue-specific or regulated promoters, inparticular from the group consisting of the CMV promoter, E1Fa promoterand metallothionein promoter. Further regulatory elements that may becontained within the vectors of this invention include, withoutlimitation, a Bcl-2 gene, UTR sequences and MAR sequences.

In a preferred embodiment, the vector is an episomic expression vector.

The above nucleic acids and vectors may be used for instance to producerecombinant mammalian IL-7 polypeptides in various competent host orhost cells, as well as for gene therapy purposes.

Another object of this invention resides in a recombinant host cellcomprising a nucleic acid or a vector as disclosed above. Such arecombinant cell may be prokaryotic or, more preferably, eukaryotic,such as a yeast, insect, plant or mammalian cell, for instance.

In a preferred embodiment, the host cell is a mammalian cell, preferablyselected from PERC6, NSO cells and BHK cells, preferably CHO cells; or ahuman cell line. The vectors, constructs and recombinant cells will bedisclosed in more details, but not limited to, in a subsequent sectionof this application.

Drug Substance and Pharmaceutical compositions

Another object of this invention resides in a drug substance comprisingas the desired product, an IL-7 polypeptide as described above,typically a hyperglycosylated IL-7 polypeptide. More preferably, thedrug substance contains less than about 10% of un- or mono-glycosylatedIL-7 polypeptide and/or is essentially devoid of product-relatedimpurities.

The invention also relates to the use of a drug substance as describedabove in the manufacture of a medicament (“drug product”) orpharmaceutical composition.

A preferred drug substance is further substantially free of processrelated impurities.

Within the context of the present application, the term “drug substance”refers to a product suitable for use as the active principle of amedicament. The “drug substance” according to this invention is, bynature, a complex product, i.e., as a result of its production method(e.g., recombinant DNA technology).

The present invention now discloses that, in order to produce efficienttherapeutic and vaccine enhancement effects, an IL-7 drug substance orpharmaceutical composition should contain, as the major molecularspecies, a hyperglycosylated IL-7 polypeptide composition.

The term “substantially free”, as used herein, indicates that the drugsubstance contains no significant or adverse amount of product-relatedimpurities and process-related impurities. More specifically, the drugsubstance should contain less than 5%, more preferably less than 3%,even more preferably less than 2% of product-related impurities andprocess-related impurities. Most preferred drug substances contain lessthan about 1% of product-related impurities and only trace amount ofprocess-related impurities.

IL-7 product-related substances designate IL-7 molecular variants, whichinclude, for example, active or inactive peptide or polypeptidefragments of IL-7.

IL-7-related impurities include, for example, human IL-7 polypeptidescomprising mono or bi-disulfide bridges, truncated IL-7, deamidatedrecombinant IL-7, dimeric or multimeric protein comprising IL-7,oxidized methionine form or a combination thereof.

Whatever their biological activity, these IL-7 molecular variants andIL-7-related impurities should be strictly minimized or discarded fromthe drug substance.

Process related impurities include, DNA, endotoxins, cell debris,viruses, etc.

A preferred drug substance is thus a drug substance wherein the totalamount by weight of a hyperglycosylated IL-7 composition comprises atleast 95% by weight, preferably at least 98% by weight, more preferablyat least 99.5% by weight of a hyperglycosylated IL-7 compositionaccording to the invention.

The invention also relates to a pharmaceutical composition comprising aneffective amount of a drug substance or hyperglycosylated IL-7composition as described above and one or more pharmaceuticallycompatible or acceptable carriers, excipients or diluents.

The invention shows that pharmaceutical compositions comprising ahyperglycosylated IL-7 composition as described above clearly increasethe vaccine properties of IL-7 and its capacity to stimulateantigen-specific immune responses.

The pharmaceutically compatible or physiologically acceptable carrier,excipient or diluent may be selected from neutral to slightly acidic,isotonic, buffered saline, solutions or suspensions and more preferablyfrom sucrose, trehalose, and amino acid. The pharmaceutically compatiblecarrier is preferably contained in an appropriate buffer to form anisotonic solution. An appropriate buffer has preferably a pH rangecomprised between 4.5 to 7.5, preferably 5.0 to 7.0, even morepreferably of about 5.5 and is preferably an organic salt selected froma sodium citrate buffer or an ammonium acetate buffer. Thepharmaceutical composition may be in the form of a suspension, solution,gel, powder, solid, etc. The composition is preferably a liquid form.

The composition may comprise stabilizing agents, such as sugar, aminoacids, proteins, surfactants, etc. The composition may comprise anysaline solution, including phosphates, chloride, etc.

A particular pharmaceutical composition according to the inventioncomprises, in addition to the active drug substance, a protein and/or asurfactant. This presence of a protein, or any other high molecularweight molecule of natural origin, reduces exposition of IL-7 to thehost immune system and therefore avoids secondary effects. Morepreferably, the protein is non immunogenic in the subject, such as anyprotein of human origin. A most preferred example of protein is humanserum albumin. The surfactant may be selected from known surfactantssuch as

Polysorbate products, preferably Tween20™ or Tween80™. A specificcomposition of this invention comprises human serum albumin (preferably2 to 5 mg/ml) or polysorbate (Tween 20 or 80 (typically 0.005%)) or anyother substance such as a tensioactive substance or amino acid (e.g.,arginine, glutamate, or a mixture of arginine and glutamate) or sugar(e.g., sucrose, trehalose, sorbitol), capable of preventing IL-7immunogenicity due to protein aggregation and/or local persistence ofthe drug product at injection site after administration of thecomposition.

In this respect, particular objects of this invention reside inpharmaceutical compositions containing a hyperglycosylated interleukin-7composition at a concentration of about 1 mg/ml to 50 mg/ml, preferablyof about 3 mg/ml to 20 mg/ml. Preferably, the effective amount ofglycosylated interleukin-7 to be administered is comprised between about10 to 200 μg/kg/week, preferably between about 10 to 60 μg/kg/week, e.g.for treatment or prevention of infectious diseases.

In view of the improved properties of the polypeptides and compositionsof this invention, the pharmaceutical compositions need to beadministered less frequently than prior art compositions or products inan equivalent amount to obtain comparable therapeutic effects. Morespecifically, in a typical embodiment, the compositions are administered3 times per week, 2 times per week, once a week, once every other week,once a month, once or twice before vaccination or before and aftervaccination. A preferred dosing regimen consists in administering thepharmaceutical composition once every 7, 10 or 14 days.

Preferred administration routes are parenteral routes. The parenteralroute is preferably an intra-tumoral, more preferably an intra-venous ora sub-cutaneous administration. It includes also intra-arterial,intra-peritoneal or intra-muscular injections. It should be understood,however, that any other suitable administration route may becontemplated depending upon the health status and the reactivity of thepatient.

In a particular embodiment, the administration route is the oral route.In comparison to other polypeptide hormones, oral route is indeedacceptable for hyperglycosylated IL-7 because of the exceptionalstability of this protein. The compositions of the invention are thenpreferably in a solid form, such as a tablet or a powder or a capsule,or in a form of a liquid, such as a syrup or an emulsion, prepared in anappropriate pharmaceutically acceptable carrier. Preferably the carrieritself is stable in the gastro-intestinal tract and in the circulatorysystem and exhibits an acceptable plasma half-life.

Gastric acid-resistant capsules, such as gastric acid-resistant capsulescontaining a micro-emulsion or liposome formulation of hyperglycosylatedIL-7 polypeptide, are advantageous.

The pharmaceutical composition may comprise additional activeingredients, such as immuno-stimulating agents, preferably selected froma hematopoietic cell growth factor, a cytokine, an antigenic molecule(or antigen) and an adjuvant, for combined, separate or sequential use.

Such additional active ingredients may be formulated in combination withthe IL-7, or, separately, for combined, separate or sequential use. In afirst variant, the active ingredients are formulated together, in thesame recipient or vessel. In another, preferred variant, they areconditioned separately, i.e., in distinct vessels or recipients.According to this embodiment, the ingredients may be administeredseparately, e.g., simultaneously or sequentially (e.g., at differentinjection sites or at different time points), to produce the mostefficient biological effect. Also, as mentioned above, repeatedadministrations of one or the two active ingredients may be performed.

In this respect, the invention relates to a pharmaceutical compositioncomprising a hyperglycosylated IL-7 composition as described above andan active ingredient selected from an immuno-stimulant and an antigenicmolecule, for combined, separate or sequential use. Adjuvants arepreferably formulated separately.

The hematopoietic cell growth factor is preferably selected from theStem Cell Factor (SCF), particularly the soluble form of the SCF, G-CSF,GM-CSF, Flt-3 ligand, IL-15 and IL-2. Typical examples of cytokines orchemokines for vaccine enhancement include cytokines that induce and/orstimulate a Th1-type immune response. The cytokine is preferablyselected from a or γ interferon, IL-2, IL-12, RANTES, B7-1, MIP-2 andMIP-1α. It should be understood that other factors such as NK cellactivators and/or NKT cell activators, FGF7 or FGF10, interleukinsand/or hormones may be used in combination with IL-7 to provideadditional therapeutic benefit.

A specific composition of this invention comprises a hyperglycosylatedIL-7 composition as described above and Stem Cell Factor, particularlythe soluble form thereof, IL-15 and/or Flt-3 ligand and/or FGF10.

Another specific composition of this invention comprises ahyperglycosylated IL-7 composition as described above and a cytokineselected from α or γ interferon, IL-2, IL-12, RANTES and MIP-1α.

Another specific composition of this invention comprises ahyperglycosylated IL-7 composition as described above, a Stem CellFactor and a cytokine.

As indicated above, the pharmaceutical composition may further compriseone or several antigens (or antigenic molecules), for combined, separateor sequential use. The antigen may be any synthetic or natural peptide,a recombinant protein, a killed, inactivated or attenuated pathogenproduct, a microorganism, a parasite, a lipid, etc., a portion thereofand a combination thereof. The antigen may be an entire protein, or anyepitope-containing fragment or portion thereof, particularly peptidesthat are presented to the immune system through MHC class I or MHC classII molecules. The antigen can be any viral antigen, bacterial antigen,parasite antigen, tumor antigen, etc. Specific examples of antigensinclude antigens derived from HIV, Varicella Zoster virus, Influenzavirus, Epstein Barr virus, type I or II Herpes Simplex virus, humancytomegalovirus, Dengue virus, Hepatitis A, B, C, D or E virus,Syncytium respiratory virus, human papilloma virus, mycobacteriumtuberculosis, Toxoplasma and Chlamydia.

A particular object of this invention relates to a compositioncomprising a hyperglycosylated IL-7 composition as described above andan antigenic molecule, for combined, separate or sequential use. Thecomposition may further comprise one or several immuno-stimulatingagents as disclosed above, for combined, separate or sequential use.

A further particular object of the present invention concerns apharmaceutical composition comprising hyperglycosylated IL-7 compositionas described above, wherein said pharmaceutical composition isadministered simultaneously, a few days before or sequentially with oneor several antigenic molecules in order to obtain and/or stimulate anantigen-specific immune response in a subject.

A further particular object of the present invention concerns a methodof causing or enhancing an antigen-specific immune response in asubject, comprising administering to a subject said antigen (or anepitope-containing fragment thereof) and a hyperglycosylated IL-7composition as described above. The composition may be administeredsimultaneously, a few days before or sequentially with, and morepreferably before said antigen in order to obtain and/or stimulate anantigen-specific immune response in a subject.

In another preferred embodiment, the composition of the inventionfurther comprises an adjuvant. The adjuvant may be selected from anysubstance, mixture, solute or composition facilitating or increasing theimmunogenicity of an antigen and able to induce a Th1-type immuneresponse, such as CpG, QS21, ISCOM and monophosphoryl lipid A. Suchadjuvants are particularly suited to produce and/or amplify a specificimmune response against antigen(s) in mammalian subjects, particularlyin humans. The adjuvant is preferably conditioned and administeredseparately from the IL-7 containing composition and/or at a distinctsite of injection, preferably with the desired antigen(s).

The present invention also concerns a pharmaceutical compositioncomprising an effective amount of a human hyperglycosylated IL-7composition according to the invention in admixture with a suitablediluent, excipient or carrier, for parenteral administration to a humanpatient for prophylactic or therapeutic stimulation of B or T lymphocytedevelopment and proliferation, or for augmentation of an immuneresponse. The pharmaceutical compositions of the invention induce aprolonged lymphopoiesis stimulation and/or amplified immune responses.

A pharmaceutical composition according to the invention may also be usedin a human patient for prophylactic or therapeutic stimulation of B or Tlymphocyte development and proliferation, for enhancement of globaland/or specific immuno-reconstitution, or for enhancement of humoraland/or cellular immune responses.

A particular pharmaceutical composition according to the invention isfor use to prevent or reduce opportunistic infections in immunodeficientpatients.

Another particular pharmaceutical composition according to the inventionis for use to prolong lymphopoiesis stimulation and/or to producespecific immune response not only against dominant epitopes but alsoagainst sub-dominant or less immunogenic epitopes, epitopes having alower affinity for the T cell receptor, which will allow to broaden therepertoire of a specific immune response in human patients.

The invention is particularly suited to produce a preventive or curativeimmune response in subjects, such as immunodeficient patients, cancerpatients, patients undergoing grafts, patients infected with a virus ora parasite, elderly patients or any patients having low CD4 count etc.

Specific and preferred uses of the IL-7 polypeptides and compositions ofthis invention include the use:

-   -   as a vaccine enhancer (administration of said composition        before, during or substantially simultaneously with antigen        administration) in an amount effective to induce enhancement of        specific immune response against malignant cells or infectious        agents; and    -   to induce immune reconstitution of patients whatever the origin:        infectious, radiations, Transplantations (BMT, SCT) or drugs;

The IL-7 polypeptides and compositions of this invention may be usedeither alone or in combination with other active ingredients, such aslymphopoietic factors including, without limitation, SCF, Flt3-L, αIFN,γIFN, IL-2, IL-3, IL-4, IL-12, IL-15, IL-18 and/or IL-21. Where combinedtherapy is used, the various ingredients may be administeredsimultaneously, separately or sequentially, and may be conditionedtogether or separately.

The IL-7 polypeptides and compositions of this invention may be used invarious areas, including to enhance vaccination in the field of animalhealth and to minimize the number of active substance administrations.

The invention further provides a method for treating a viral infection,such as HIV infection, viral hepatitis, West Nile fever, Dengue, whichmethod comprises administering to an infected patient, ahyperglycosylated IL-7 polypeptide composition.

In a particular embodiment, the hyperglycosylated IL-7 polypeptide is tobe administered in association with an interferon molecule. Theinterferon molecule can be for instance alpha IFN (leukocyte IFN), betaIFN (fibroblast IFN), gamma IFN (immune IFN), omega IFN or tau IFN(trophoblastic factor).

The invention further provides a method for improving a thymopoieticrecovery in immuno-compromised subject, which method comprisesadministering to an immuno-compromised subject, a hyperglycosylated IL-7polypeptide composition.

Preferably the hyperglycosylated IL-7 polypeptide is then to beadministered in association with a keratinocyte growth factor, a stemcell factor, a gonadostimulin antagonist or a growth hormone.

The hyperglycosyltated IL-7 polypeptide can also be used in a method forproviding a therapeutic immunization against malignant cells, virus orbacteria, wherein the hyperglycosylated IL-7 polypeptide is to beadministered in association with an antigen or a mixture of antigens,e.g. those described above. In this situation, the hyperglycosylatedIL-7 polypeptide may be further administered in association with GM-CSF.

A further object of this invention is a method for ex-vivo enhancingexpansion of T cells, which method comprises contacting T cells with ahyperglycosylated IL-7 polypeptide or composition, hereby enhancingexpansion of the T cells. This method is particularly useful to prepareT cells suitable for treating patients with cancer or viral infection byadoptive immunotherapy. Adoptive immunotherapy is an ex vivo methodologyfor selective expansion of specific T cells targeting specific antigens(malignant or viral). This immunotherapeutic technique generallyincludes isolation of Ag-specific T lymphocytes from whole blood of thepatient, ex vivo expansion of theses T cells using IL-7 polypeptides,optionally ex vivo activation of theses T cells by other cytokines andadministration to the patient. Other techniques are possible. IL-7polypeptides improve survival of these T cell populations which furthershow an enhanced cytotoxic activity.

Production Methods and Tools

Another aspect of the present invention is to provide appropriateconstructs and methods for producing the above compositions,particularly the above hyperglycosylated IL-7 polypeptides, compositionsand drug substances, in sufficient quantities and quality forpharmaceutical use thereof.

In particular, as discussed above, the present invention providesvectors as well as recombinant host cells that may be used to producerecombinant human IL-7 polypeptides of this invention in variouscompetent host cells, as well as for gene therapy purposes.

The vector may be a plasmid, virus, phage, cosmid, episome, etc.Preferred vectors are viral vectors (e.g., recombinant adenoviruses) andplasmids, which can be produced based on commercially availablebackbones, such as pBR, pcDNA, pUC, pET, pVITRO, etc. The vectortypically comprises regulatory elements or sequences to control ormediate expression of an IL-7 polypeptide. The regulatory sequences maybe chosen from promoters, enhancers, silencers, tissue-specific signals,peptide signals, introns, terminators, polyA sequences, GC regions,etc., or a combination thereof. Such regulatory elements or sequencesmay be derived from mammalian, fungal, plant, bacterial, yeast,bacteriophage or viral genes, or from artificial sources. Usefulpromoters for prokaryote expression (such as E. coli) include T7 RNApolymerase promoter (pT7), TAC promoter (pTAC), Trp promoter, Lacpromoter, Tre promoter, PhoA promoter for example. Suitable promotersfor expression in mammalian cells include viral promoters (e.g., CMV,LTR, RSV, SV40, TK, pCAG, etc.), domestic gene promoters (e.g., E1fα,chicken βactine, Ubiquitine, INSM1, etc.), hybrid promoters (e.g.,actine/globin, etc.), etc. A vector may comprise more than one promoter.The promoters may be inducible or regulated. For instance, the use ofinducible or regulated promoters allows a better control of productionby dissociating the culture from production phases. Inducible orregulated promoters may be found in the literature, such as theTetracycline system, the Geneswitch system, the Ecdysone system, theOestradiol system, the RU486 system, the Cumate system, themethallothioneine promoter etc. Other systems are based on electriccurrents or microwaves, such as focalized ultrasound system, AIR inducedexpression system and the like. These systems can be used to controlexpression of an IL-7 polypeptide according to the invention.

The IL-7 may be co-expressed with an anti-apoptotic factor (e.g., iex,Bcl2, Bc1XL, etc.) or cycline (e.i. p21, p27, etc.). The eDNAs codingfor said IL-7 and for said anti-apoptotic factor may be both placeddownstream of the same promoter, but separated by an IRES sequence, oreach of them downstream of its own promoter.

The vector may further comprise an origin of replication and/or a markergene, which may be selected from conventional sequences. Anamplification selection marker such as the DHFR gene can be inserted inthe backbone of the vector.

The vector may further comprise various combinations of these differentelements which may be organized in different ways.

The present invention also provides recombinant host cells comprising anucleic acid or a vector as described above. The host cell may beselected from any eukaryotic and prokaryotic cells, typically from amammalian cell (in particular a human, rodent, canine cell), a bacterialcell (in particular E. coli, Bacillus Brevis, Bacillus Subtilis), ayeast cell, a plant cell and an insect cell. These host cells may beadapted to serum-free media. Production may also be accomplished in atransgenic animal or plant.

Preferred recombinant host cells are selected from mammalian cells, inparticular human cells as well as derivatives or mutants thereof.

Specific examples of suitable host cells include Chinese Hamster Ovary(CHO) cells, Baby Hamster Kidney (BHK) cells, Human Embryonic Kidney(HEK-293) cells, human epidermal keratinocytes (HEK), human stromal orepithelial cells, PERC6, etc. In such mammalian cells, IL-7 may beproduced as a secreted protein using functional signal peptidesequences.

A specific object of this invention is a eukaryotic host cell comprisinga nucleic acid molecule comprising SEQ ID NO: 2, 4 or 6.

A further object of the present invention relates to antibodiesimmunoreactive with an IL-7 composition or polypeptide as describedabove. Such antibodies may be produced according to conventionalmethods, including immunization of animals and collecting the serum(polyclonal) or preparing hybridomas from spleen cells (monoclonal).Fragments (e.g., Fab') or engineered derivatives of antibodies (e.g.,ScFv or diabodies or minibodies) may be produced by known biological andchemical methods. Preferred antibodies are specifically immunoreactivewith a hyperglycosylated IL-7 polypeptide as described above, i.e., canbind the hyperglycosylated IL-7 polypeptide without substantiallybinding un- or mono-glycosylated polypeptides. Although non-specific orless effective binding to such other antigens may be observed, suchnon-specific binding can be distinguished from specific binding to theparticular hyperglycosylated IL-7 polypeptides of this invention.

The antibody is preferably of simian, murine or human origin or has beenhumanized.

The invention also relates to a hybridoma cell line that produces amonoclonal antibody as described above.

Such antibodies are useful in detecting hyperglycosylated IL-7polypeptide or in neutralizing IL-7 biological activity in assays orexperiments involving multiple lymphokines. A composition suitable fordiagnosis, assay or therapy comprising such monoclonal antibodies isalso an object of the present invention.

Another object of the present invention relates to processes which canbe used, on an industrial scale, for the production of a pharmaceuticalgrade, substantially pure hyperglycosylated IL-7 polypeptide asdescribed above. The process leads to high yields of recombinant IL-7conformer suitable for therapeutic use. The invention also providesnovel methods of controlling IL-7-containing compositions, to determinethe presence of amount of hyperglycosylated IL-7 polypeptide asdescribed above.

In a particular aspect, the method of producing hyperglycosylated IL-7polypeptides or compositions as defined above comprises:

a) culturing a recombinant host cell as described above, and

b) collecting an IL-7 polypeptide produced from said cells.

The sample may be subjected to various treatments or conditions in orderto increase purity of IL-7, to remove cell debris or viral particles,etc. Typical examples of such treatments include centrifugation,clarification and/or dia-ultra-nano-filtration. The sample may thus beenriched for IL-7 polypeptide.

To increase the yields or efficiency of the method, it is highlydesirable to produce a sample containing or enriched in correctly foldedand glycosylated IL-7 polypeptides.

The hyperglycosylated IL-7 polypeptide may be purified by differenttechniques known per se, but which have not been used so far in thepresent combination to produce a hyperglycosylated IL-7 polypeptide.These techniques are more preferably selected from hydrophobicinteraction chromatography, ion exchange chromatography, affinitychromatography and gel filtration chromatography, either alone or invarious combinations. Such methods allow removal of host cell DNA andother impurities which would lower recovery. In a preferred embodiment,step ii) comprises a hydrophobic interaction chromatography step. Suchchromatography may be carried out using various supports and formats,preferably using HIC butyl. Step ii) may be carried out on any support,preferably on batch or in column using an appropriate gel.

In a preferred embodiment, the purification step comprises loading thesample through a column packed with a specific gel (Sephadex forexample).

In another preferred embodiment, the purification step comprises apolishing step involving loading the sample through a column packed witha specific gel (Source 15S) to concentrate recovered protein of interestand eliminate possible residual protein contaminants.

In another particular embodiment the purifying step comprises loadingthe sample through a column packed with a specific gel comprising amonoclonal anti IL-7 antibody immobilized on a resin (dextran sulfate orheparin for example).

These methods allow the reproducible and efficient production of asubstantially pure hyperglycosylated IL-7 polypeptide as describedabove. The methods are particularly advantageous since the recombinantIL-7 can be obtained with a purity of at least 95% by weight, preferablyat least 98% by weight and even more preferably at least 99% or even99.5% by weight with respect to the total amount of IL-7.

Each step of the above described process may be controlled by analyticalmethods, including SDS-PAGE analysis. The primary structure of theoptimized IL-7 may be controlled and characterized by determining thegene and/or amino acid sequence, by peptide mapping analysis, aftertrypsic digestion, by determining molecular weight with SDS PAGE, sizeexclusion HPLC, Mass spectrometry such as MALDI TOF or electrospray orthe like, by determining hydrophobicity with reverse phase HPLC forexample, and/or by determining the electric charge with cation exchangechromatography HPLC or isoelectrofocalisation analysis for example.

A further embodiment of the invention relates to IL-7 production methodsas described above, wherein IL-7 expression by the recombinant hostcells is inducible, regulated or transient, so that the cell culture andIL-7 expression phases can be dissociated. More particularly, in aparticular embodiment, IL-7 expression can be repressed or minimizedduring recombinant cell growth, expansion and/or culturing, to allow theproduction of large amounts of recombinant host cells without anyIL-7-mediated potential toxic effect. Then, IL-7 expression can beinduced within the cell culture (or on a sample thereof), allowing theefficient synthesis and release of recombinant IL-7.

An object of this invention thus also resides in a method of producing arecombinant IL-7 polypeptide, comprising culturing a recombinant hostcell as disclosed above comprising a nucleic acid molecule encoding saidIL-7 polypeptide and recovering the recombinant IL-7 polypeptideproduced, wherein said nucleic acid molecule provides for a regulated orinducible expression of said IL-7 polypeptide, so that expression ofsaid IL-7 polypeptide can be repressed or minimized during recombinantcell growth and induced during production phase. The nucleic acidtypically comprises an inducible promoter, which can be repressed oractivated in the presence or absence of a specific agent contained oradded into the culture media. The method is particularly suited toproduce an IL-7 hyperglycosylated conformer as disclosed above.

Various regulated or inducible expression systems have been disclosed inthe art, which are functional in mammalian host cells and can be used inthe present invention. These include the Tetracycline TetOn/Off system,Geneswitch system (Invitrogen) with Mifepristone as inducible agent andGAL4-E1b promoter, Ecdysone system (induction with ponasterone A ormuristerone A, analogs of insect steroid hormones) (Invitrogen),methallothioneine promoter (inducible by zinc), Oestradiol system, RU486system, focalized ultrasound system, AIR (Acetaldehyde inducibleregulation) induced expression system, Cumate system (Q-mate; Qbiogen),Cre-Lox system, etc. These regulated or inducible expression systems maybe used in various cells, such as for instance HEK293, HEK293 EBNA, HEK,T-REX™-293, T-REX™-HeLa, T-REX™-CHO or T-REX™-Jurkat cell lines,transformed with a recombinant vector designed to express recombinantIL-7 after induction.

Alternatively, transient transfection can be used to dissociate cellexpansion from IL-7 production. In this regard, efficient gene deliveryvectors are used to introduce an IL-7 coding sequence into cells uponexpansion thereof. More preferably, the vector system for transienttransfection is a viral vector, such as a recombinant adenovirus or anepisomal vector [e.g., pCEPH (Invitrogene), pTT (IRB: Durocher Y. et al.Nucl. Acids. Res., 2002, 30(2)) or using MAR sequences]. Adenoviruses(and other viral vectors such as AAVs, for instance), can be producedaccording to techniques known in the art. Typically, E1-defectiveadenoviruses are produced in a E1-complementing cell line, such asHEK293, PERC6 cells, etc. Such transient transfection process can beimplemented in various mammalian cells in culture, such as A549-, HeLa-,VERO-, BHK- or CHO-transformed cells for example (as disclosed inexample A4). An alternative transient expression method suitable for usein the present invention is disclosed for instance in the next article:Durocher Y. et al. Nucl. Acids. Res., 2002, 30(2) in HEK293 EBNA orHEK293 cells.

In a preferred embodiment, the production methods of this inventioncomprise an additional step c) of characterizing and measuring orquantifying the particular hyperglycosylated IL-7 polypeptide asdisclosed above contained in the resulting product. The physical andbiological characterization of the desired hyperglycosylated IL-7polypeptide may be obtained by Mass spectrometry (MALDI-TOF orelectrospray), infra-red spectroscopy, nuclear magnetic resonance (NMR),by determining circular dichroism, by assessment of the biologicalactivity of the IL-7 in a specific bioassay, by measuring the affinitytowards a specific monoclonal antibody raised against saidhyperglycosylated IL-7 polypeptide, or heparin affinity HPLC. Oncecharacterized, the quantification of said conformer may be performed byELISA, bioassay, affinity of said hyperglycosylated IL-7 polypeptide forIL-7 receptor and any method of protein quantification if applied to theisolated conformer.

In this regard, the invention also provides and concerns a method toidentify and/or measure the quantity of hyperglycosylated IL-7polypeptide and/or related impurities in a sample, particularly in apharmaceutical preparation. Such characterization methods can be used toinitially characterize and qualify the protein for filing a therapeuticuse, in quality control of pharmaceutical batches. The inventionproposes, for the first time, a method of characterizing and controllingIL-7-containing preparations, to determine the presence and/or relativequantity of hyperglycosylated IL-7 polypeptide of this invention.Preferred methods use Bicinchoninic Acid (BCA) protein Assay, SDS-PAGE,western blot, size-exclusion HPLC, reverse phase HPLC, ion exchangeHPLC, hydrophobic interaction HPLC, Amino Acid Assay (AAA),Isoelectrofocalisation (IEF), ELISA, UV absorption and/or a Bioassay.These methods may be carried out alone or in various combinations.

The invention also provides a method of producing an IL-7 drug substanceor pharmaceutical composition, said method comprising (i) culturing arecombinant host cell encoding an IL-7 polypeptide, (ii) isolating saidrecombinant polypeptide to produce an IL-7 drug substance and (iii)conditioning said IL-7 drug substance to produce a pharmaceuticalcomposition suitable for therapeutic or vaccine use, said method furthercomprising a step of identifying, characterizing or measuring, in saiddrug substance or pharmaceutical composition, the quantity and/orquality of hyperglycosylated IL-7 polypeptide as defined above and, morepreferably, a step of selecting the drug substance or pharmaceuticalcomposition which comprises, as the active ingredient, more than about90%, preferably 95%, more preferably 98% of said hyperglycosylated IL-7polypeptide.

The characterizing step may be carried out by a variety of techniques,more preferably by mass spectrometry-related methods, with or withouttrypsic digest, Lectine Affinity Chromatography, Amini Acid Assay (AAA),Endo- and Exo-N- and O-glycanase digestions (PNGase A/F, O-glycosidase,neuraminidase), Fluorophore Assisted Carbohydrate Electrophoresis, MALDITOF or Electrospray Mass Spectrometry, specific monoclonal antibodyanalysis for disulfide bridges and/or conformation characterization. Theidentification of molecular variants and product-related impurities ispreferably performed by using one or several methods selected frombi-dimensional electrophoresis, isoelectric focusing and ion-exchangechromatography for deamidated forms, size exclusion chromatography andSDS-PAGE analysis for multimeric forms, and HPLC reverse phase with orwithout enzymatic predigestion for truncated forms.

The step is particularly suited for quality control of clinical orpharmaceutical compositions, whereby only compositions comprising morethan about 95% of the above hyperglycosylated IL-7 polypeptide areretained, preferably more than about 96%, 98% or 99.5%. All thesehyperglycosylated IL-7 polypeptide showing an average isoelectric pointbelow 6.5.

Another object of the present invention relates to the use of arecombinant hyperglycosylated IL-7 polypeptide obtained with theprocesses as described above, for the manufacture of a pharmaceuticalcomposition to prevent or treat a disease associated with animmunodeficiency, particularly to induce a prolonged lymphopoiesisstimulation, to cause and/or amplify an immune response, particularly anantigen-specific immune response.

A further object of the invention relates to the use of ahyperglycosylated IL-7 polypeptide as a tool for experimental andpharmacological use in mammalians for veterinary applications.

Other aspects and advantages of the present invention will be describedin the following examples, which should be regarded as illustrative andnot limiting the scope of the present application.

EXAMPLES Example A Construction and Expression of Optimized Human (H)and Simian (S) IL-7-Coding Nucleotide Sequences in Mammalian Cells A1.Construction of an Optimized Human IL-7-Coding Nucleotide Sequence:

1.1. Peptide Signal Optimization:

As the expression of IL-7 cDNA fragments linked at the 5′ end to thenatural IL-7 peptide signal was very low, we tested several signalpeptide sequences.

The new human IL-7 encoding cDNA sequences were chemically obtained fromassembled synthetic oligonucleotides.

Several signal peptide sequences were tested: signal peptide (SP) ofhighly secreted proteins (Barash et al.; 2002; Biochemical andBiophysical Research Communications 294:835-842):

IL-7 SP MFHVSFRYIF GLPPLILVLL PVASS (SEQ ID NO: 13) EPO SP MGVHECPAWLWLLLSLLSLP LGLPVLG (SEQ ID NO: 14) SEAP SP MLLLLLLLGL RLQLSLG (SEQ IDNO: 15) IgGkappa SP METDTLLLWV LLLWVPGSTG (SEQ ID NO: 16)Lactotransferin/vitronectin SP MKLVFLVLLF LGALGVALA (SEQ ID NO: 17)Cystatin bis SP MARPLCTLLL LMATLAVALA (SEQ ID NO: 18) EPO/IL-7 a newhydrid SP MGVHECPAWL WLLLSLLSLV LLPVAS (SEQ ID NO: 19)

The obtained cDNAs sequences were inserted into the pTT5 vector(Durocher et al.; 2002; Nucl. Ac. Res.; 30) for transient expression inmammalian cells such as HEK293 cells, CHO cells.

To check for the good cleavage of the signal peptide, the N terminalamino acid was determined for each obtained protein; hIL-7 integrity wasmaintained.

Cystatin, IgG, EPO and the hybrid EP/7 appeared as the best signalpeptide sequences. Indeed, hIL-7 expression is enhanced by, at least, afactor 10.

1.2. Human IL-7-Coding Nucleotide Sequence Optimization:

Maintaining the EP/7hIL-7 amino acid sequence, the nucleic acid sequencewas optimized by

-   -   elimination of human rare codons (using Graphical Codon Usage        Analyser software)    -   enhancing mRNA stability by enhancing “GC” content of the        sequence, except for the signal peptide sequence (Kim et al.;        1997; Gene; 199), and minimizing succession of “CA”        dinucleotides.

The sequence is depicted in SEQ ID NO: 2.

A2. Construction of an Optimized Simian IL-7-Coding Nucleotide Sequence:

As described for the human IL-7-coding sequence, an EP/7-sIL-7 optimizedsequence was synthesized (SEQ ID NO: 3).

A3. Construction of Canine IL-7-Coding Nucleotide Sequence:

Canine IL-7 cDNA was amplified by PCR from a dog kidney cDNA library(Biochain), cloned and sequenced as described above for the humanIL-7-coding sequence, an IL-7SP or EP/7SP-cIL-7 sequence was synthesized(SEQ ID NO: 6).

A4. Mammalian Expression (BHK Cell Expression, or CHO Cell Expression orHEK-293 Cell Expression):

The IL-7 encoding cDNA sequences were amplificated by polymerase chainreaction (PCR) (Mullis et al.; 1987; Methods in Enzymology; 155:335-350)to create the restriction sites (NotI/SwaI) for cloning into theexpression vector.

The expression system ph-pgk.EP7-hIL-7 (FIG. 1) or pBh-pgk.EP7-hIL-7(FIG. 2) was designed to express an IL-7 protein predicted from thetranslation of the natural human IL-7 gene sequence. Selection forrecombinant vector-containing cells was doned on the basis of theantibiotic (Ampicilin for cloning in E. coli and hygromycin forexpression in mammalian cells) resistance marker genes carried on thevector.

This expression vector has been entirely constructed at CYTHERIS,beginning from pIC20H plasmid (ATCC) conferring ampicillin resistance tothe system. It contains 2 mammalian production units:

1/one for the expression of the IL-7 encoding sequences, under thecontrol of the pgk promoter, and a synthetic polyA sequence avoidingtranscription through the pgk promoter.

2/one for the expression of the hygromycin resistance, under the controlof the “sv40 enhancer tk promoter”.

Following sequences were inserted into this preliminary vector:

-   -   “hph-ef1a pA”: HindIII/SbfI fragment from pVitro2.mcs        (Invitrogen);    -   tk promoter: EcoRI/HindIII PCR fragment from pMEP4 (Invitrogen);    -   sv40 enhancer: BssHII/EcoRI PCR fragment from pVitro2.mcs        (Invitrogen);    -   MAR rabbit Bglobin: a putative “Matrix Attachment Region” for a        better integration in highly transcribed region of the        chromatin, EcoRV/AgeI rabbit βglobin intron2 PCR fragment from        pSG5 (Stratagene);    -   SpA: StuI/BspEI fragment from pCAT3 control (Promega)    -   Pgk promoter: KpnI/BssHII PCR fragment from pQBI.pgk (Q-biogen);    -   5′UTRint1: HindIII chimeric intron fragment from pCAT3-control        (Promega);    -   NotI/SwaI or NotI/PmlI IL-7 encoding cDNA and mutants;    -   hghpA: NruI/SwaI synthetic synthesis from human growth hormone        cDNA sequence described by M. Goodman (DeNoto et al.; 1981;        Nucl. Acid. Res.; 9 (51):3719-3730).

Some variants of the vector were prepared with other IL-7 promoter thanthe pgk promoter: Ef1alpha, snRNA U1, actin, Ubiquitin, CMV promoters,etc, or other selection marker: neomycine, etc.

The mammalian (HEK-293, CHO or BHK) expression vector comprising SEQ IDNO: 2, is called ph-pgk.EP7-hIL-7 or pBh-pgk.EP7-hIL-7. StableExpression of human IL-7 in HEK-293 or CHO transfected cells wasachieved using the expression vector ph-pgk.EP7-hIL-7 orpBh-pgk.EP7-hIL-7. After linearization by NdeI, expression vector,ph-pgk.EP7-hIL-7 or pBh-pgk.EP7-hIL-7, was transfected in the mammalianhost cells using methods known to those skilled in the art. Theselectable marker used to establish stable transformants was hygromycin(Invitrogen).

A5. Inducible Mammalian Expression (Methalothioneine Promoter “MT1”):

In the same expression vector, pgk promoter has also been replaced by achemically synthesized BspEI/BssHII “Mus musculus MT1” sequence, asreferred in PubMed (No X53530) (Carter et al.; 1984; Proc. Natl. Acad.Sci. USA; 81:7392-7396).

MT1 is a metal-dependent transcription factor promoter. Expression ofstable clones is then zinc dependent.

A6. Mammalian Co-Expression of IL-7 and Bc12 or Bc1XL (BHK CellExpression, or CHO Cell Expression or HEK-293 Cell Expression):

In order to enhance cell viability in mammalian host cell culture andtherefore to optimize the amounts of IL-7 production, a variantexpression plasmid was prepared by inserting Bc12 cDNA sequence inbetween tk promoter and hph cDNA so that anti apoptotic action of Bc12could be tested in bioreactor production (Zhong et al.; 1993; Proc.Natl. Acad. Sci. USA; 90:4533-4537-Lee et al.; 2000; Journal of cellScience; 114(4):677-684). (See FIG. 2).

Example B Construction and Expression of Hyperglycosylated Analogs ofIL-7-Coding Nucleotide Sequences in Mammalian Cells

B1. Construction of cDNA Sequences of Hyperglycosylated IL-7 Analogs

The hyperglycosylated IL-7 analogs were obtained using severaltechniques including mutagenesis methods. Hyperglycosylated IL-7 analogs(alternatively: HG37-40-104-126 and -147) were chemically constructedfrom assembled synthetic oligonucleotides. Several analogs were obtainedby introducing one or more desired mutations so that giving IL-7 analogshaving one or more additional glycosylation sites. Thus the resultingfull length cDNA sequence containing one or multiple desired additionalglycosylation sites were inserted, after digestion with NotI and PmlIrestriction enzymes, in between the NotI/PmlI restriction sites fordirect cloning into the expression vector (similar to FIG. 1 or 2 butcontaining appropriate IL-7 sequence).

B2. Expression of cDNA Sequences of Hyperglycosylated IL-7 Analogs

The expression of hyperglycosylated IL-7 analogs was conducted asdescribed above in sections A4 to A6.

Example C Production of Recombinant HIL-7 in Bioreactor CultureConditions

The best stable positive clone, as in example A4 was adapted toserum-free suspension culture by several media and components screeningsin order to produce a clone optimized for productivity and growth inhigh cell density culture. Before seeding the 100 to 2000 L bioreactor,pre-cultures are performed in the “wave bag” system. Cell culture isperformed in a 100 to 2000 liters bioreactor with a perfusion system ora fed-batch system during 10 to 15 days. Cells were amplified to aconcentration of 10 millions cells/ml in a low-glutamine content mediumsupplemented with plant peptones.

In a first expansion step the culture temperature is regulated at 37° C.to increase cell density. After a few days, the temperature was loweredat around 28/32° C. to inhibit cell growth and allow a better expressionlevel. Moreover, decreasing temperature decreases the speed of thesecretion pathway, favoring better glycosylation of the expressed IL-7with increased site occupancy.

A few days before the end of the culture, IL-7 expression was boosted byaddition of 0.5-10 mM Sodium Butyrate in the medium.

Under conditions described above, IL-7 expression was monitored bothinside the cells and in the culture medium (FIG. 3).

To produce high MW IL7 glycoforms, 3 g/L glucose and 3 mM glutamine aremaintained in the medium during the culture as well as a goodoxygenation. One also monitors the amino acids consumption and feeds theculture with depleted amino acids. Cell culture is harvested as soon ascell viability decreases below 90%.

Example D Purification of Recombinant Human IL-7 Product Expressed inHEK-293 and CHO Cells

Crude cell culture medium was collected and centrifuged to pellet wholecells and cells debris. Alternatively this can be achieved by in depthfiltration on clarification capsules or modules such as Mustang XTcapsule (Pall), Sartoclear P (Sartorius), Millistak+Opticap (Millipore)or hollow fiber cartridges (AXH cross flow 10 (GE)) or equivalent.Centrifuged culture medium was concentrated approximately 10-fold withCentrasette Cassette apparatus, membrane cut off 10 kDa (Pall LifeSciences) to reduce the volume of supernatant. Any otherfiltration/concentration system with similar porosity could also beused.

The concentrated supernatant was centrifuged, adjusted to pH 7.5 andapplied to a Q Sepharose Fast Flow (General Electric Healthcare) columnequilibrated with 50 mM sodium phosphate pH 7.5. The protein was thenrecovered in the flow through. During this negative chromatographicstep, various contaminants among which DNA were eliminated. Analternative to this step was to use validated Mustang Q membranecassettes (Pall) in similar conditions, for better yield and/or slightlyfaster process. Another alternative to this step is to capture theprotein on a strong Anion exchanger resin (Q Ceramic Hyper D (Biosepra),Capto Q (GE)) or membrane (Sartobind Q, Sartorius).

After this prepurification step, a capture step was performed on astrong cation exchanger resin. The flow through collected at the end ofprevious step was loaded onto a Fractogel EMD SO3⁻ (Merck) columnequilibrated with loading buffer (50 mM sodium phosphate pH 7.5), andwashed with 50 mM sodium phosphate pH 7.5. Elution was carried out usinga linear NaCl gradient (15 column volumes) in 50 mM sodium phosphate pH7.5.

Active fractions were pooled and inactivated during 30 minutes at pH 3.5at room temperature to eliminate virus. An alternative to this processis to replace this viral inactivation step by a multilayernanofiltration at the end of the process.

After viral inactivation, pooled protein fractions were diluted 2-foldin buffer (200 mM sodium phosphate pH 7, 3M ammonium sulphate) and pHwas adjusted to 7. Then, the protein solution was loaded onto aHydrophobic Interaction Chromatography (HIC) Butyl Toyopearl 650-M(Tosoh) column equilibrated with the loading buffer (50 mM sodiumphosphate pH 7+1.5M ammonium sulphate). After washing with the loadingbuffer, IL-7 was eluted with 25 column volumes of a salt gradientranging from 1.5 M to 0 M ammonium sulphate in 50 mM sodium phosphate pH7.

Alternative HIC resin such as hexyl Toyopearl 650-M (Tosoh), Butyl/OctylSepharose™ 4 Fast Flow (General Electric Healthcare), can be utilizedfor this step, Another alternative to HIC for scaling up purposes was touse another matrix such as MEP HyperCel (Pall Biosepra) for similarresults.

The combination of the above-mentioned capture step and HydrophobicInteraction Chromatography allowed optimal separation of the differentglycosylated IL-7 isoforms (from B1 to B10 as indicated on FIG. 4),according to their intrinsic physico-chemical properties. Adequateselection of elution fractions (fraction from B1 to B4) lead to anenrichment in the 3N-associated or not to 1O-glycosylated hIL-7 entity.An example of such glycoform separation is shown in FIG. 4.

The highly glycosylated IL-7 fractions were pooled and loaded onto a G25Sephadex (General Electric Healthcare) column equilibrated with low saltbuffer (20 mM sodium acetate pH 6). An alternative to this step is todiafiltrate the high salt protein pool using 5 or 10 KDa molecularweight cut off TFF membranes (Qvick start membranes, (GE), CentramateTFF (Pall)).

The protein fractions obtained from G25 step were loaded onto a Source15S (General Electric Healthcare) column equilibrated with the loadingbuffer (20 mM acetate sodium pH 6). This polishing step resulted inprotein concentration and elimination of the residual contaminants.

The column was washed with sodium acetate loading buffer and the IL-7protein was eluted with 15 column volumes of a salt gradient rangingfrom 0 to 1 M NaCl in 20 mM sodium acetate pH 6. Eluted fractions wereseparated by SDS-PAGE and stained with either Coomassie blue or silverNitrate. Only the fractions containing IL-7 were pooled to release thefinal purified IL-7 protein batch.

If viral inactivation has not been conducted before, purificationprocess may also include an additional combination of two filtrations toguaranty optimal viral clearance. Viral removal can be achieved byfiltration using a prefiltration device (Planova 75, Asahi KaseiMedical) followed by a nanoporous cellulose membranes (Planova 20N,Asahi Kasei Medical) or by other viral removal membranes (Virosart,Sartorius; DV20, Millipore).

SDS PAGE of the purified E. coli, glycosylated and hyperglosylated hIL-7are shown on FIG. 5.

Shifts in the gel illustrate the level of glycosylation of the protein.Indeed, the hyperglycosylated forms tested here (HG-37-147 andHG-40-104) have a higher molecular weight than the full glycosylatedhIL-7.

Example E Analysis of Glycoprotein Carbohydrates

Production of recombinant human IL-7 was conducted in a CHO cell-basedexpression system for, but not limited to, the following reasons. CHOcells are the current most validated and most common host used for theproduction of recombinant human therapeutic glycoprotein. Furthermore, alarge set of detailed work reported that CHO cells, includinggenetically modified CHO cell lines expressing sialyl-α-1-6 transferase,were able to glycosylate recombinant proteins in a manner qualitativelysimilar to that observed in human cells. This particular feature was ofmajor importance to reduce the potential immunogenicity of therecombinant glycoprotein when injected to human patients.

Purified recombinant human IL-7 product or fractions enriched forparticular glycoforms (3N or 3N+2N, associated or not to 1 O-glycanmoiety) obtained from transfected CHO cells were analysed by westernblot to confirm glycosylation status in comparison to E. coli-derivedrecombinant human IL-7.

The different glycoforms of the CHO-produced and purified IL-7 weredifferentially characterized using PolyAcrilamide Gel Electrophoresis.Apparent molecular weight glycoprotein entities were ranging between 20KDa and 35 KDa with a major band at around 27 KDa (observed in SDS-PAGE,see FIG. 5 and FIG. 6), most probably corresponding to a threeN-glycosylated form comprising or not one O-glycan moiety. This aspectwas specifically addressed by enzymatic deglycosylation of the purifiedproduct (FIG. 7).

These glycoforms (3N or 3N+2N, associated or not to 1 O-glycan moiety)of the CHO-produced and purified IL-7 were differentially characterizedusing mass spectrometry, giving molecular mass superior to 25 KDa forthe 3N-glycoform associated or not to 1 O-glycan moiety and superior to23 KDa for the 2N-glycoform associated or not to 1 O-glycan moiety (seeFIG. 8).

Furthermore, the above glycosylated forms present an average isoelectricpoint of 5.8 reflecting a high sialylation profile (see FIG. 9).

As a comparison, similar analysis with unglycosylated E coli-derivedhIL-7 gave a protein with an apparent molecular weight at approximately18 KDa, and mammalian cells derived hyperglycosylated hIL-7 wereexhibiting apparent molecular weight comprised between 27 and 37 KDa

General glycosylation complexity and total N-glycan heterogeneity of thepurified CHO-derived hIL-7 was assessed by total enzymaticde-glycosylation followed by chromatography separation and massspectrometry analyses of the generated oligosaccharides.

Purified glycosylated h-IL-7 samples were enzymatically digested with anendoglycosidase such as peptide-N-glycosidase F (PNGaseF, Roche).Released N-linked oligosaccharides were separated from the peptidestructure and sorted using a graphite Carbograph 200-300 μl column(Alltech), followed by MALDI-TOF Mass Spectrometry (Voyager Spec,Applied Biosystems). The m/z values corresponding to each peack of theMS spectrum allowed identification of the N-Glycan general structure ofthe whole hIL-7 molecule.

For specific detection of the sialic acid containing glycans, acarboxymethylation of the PNGase-generated oligosaccharides (as reportedin Powell A K & Harvey D J, Rap. Corn. Mass Spec. 1996) was undertakenprior to Mass spectrometry analysis.

Analysis of the spectrum generated from purified CHO-derived hIL-7revealed N-glycans masses ranging from 1340 Da up to 3516 Da. (See FIG.10).

From the spectrum, the following glycan structure could be determined(see Table 3):

TABLE 3 m/z Signal Assignment of observed molecular ions 1338Hex₃HexNAc₄ + Na⁺ 1448 Hex₄(dHex)HexNAc₄ + Na⁺ 1485 Hex₃(dHex)HexNAc₄ +Na⁺ 1647 Hex₄(dHex)HexNAc₄ + Na⁺ 1809 Hex₅(dHex)HexNAc₄ + Na⁺ 1824Hex₃(dHex₂)HexNAc₅ + Na⁺ 1970 Hex₅(dHex)HexNAc₄(Sulph)₂ + 2Na⁺ 2012Hex₅(dHex)HexNAc₅ + Na⁺ 2157 NeuAcCarboxyHex₄(dHex)HexNAc₅ + Na⁺ 2182NeuAcHex₅(dHex)HexNAc₄Sulph + Na⁺ 2318 NeuAcCarboxyHex₅(dHex)HexNAc₅ +Na⁺ 2421 Hex₃(dHex)HexNAc₈(Sulph) + Na⁺ 2536 NeuAcCarboxyHex₆HexNAc₆ +Na⁺ 2624 NeuAc2CarboxyHex₅(dHex)HexNAc₅ + Na⁺ 2786NeuAc2CarboxyHex₆(dHex)HexNAc₅ + Na⁺ 2843 NeuAc2CarboxyHex₆HexNAc₆ + Na⁺3092 NeuAc3CarboxyHex₆(dHex)HexNAc₅ + Na⁺ 3153NeuAc3CarboxyHex₆HexNAc₆ + Na⁺ Hex: hexose (Galactose or Mannose),HexNAc: N-acetylhexosamine (N-acetyl Glucosamine or N-acetylGalactosamine), dHex: deoxyhexose (Fucose), Sulph: Sulfate group, NeuAc:N-acetyl neuraminic acid

Taking into account i) the respective masses of the observedoligosaccharide moieties, ii) the mass of each monosaccharide and iii)laws of glycan biosynthesis pathways as they are known today, thefollowing highly complex N-glycan structures can be assumed with goodprobability.

TABLE 4 Complex bi and triantennary mammalian N-glycans characterized onCHO-derived hIL-7(but not limited to): 2420

2477

2536

2623

2785

2843

3092

3149

◯ Mannose □ N-acetylglucosamine  Galactose ⋄ α 1-3-Fucose Δ Sialic Acid

Glycosylation complexity was also assessed via determination of molarratio of the different monosaccharides found on all the glycans (N- andO-glycan if applicable) of the purified CHO-derived hIL-7.

All the glycans of purified glycosylated h-IL-7 samples were chemicallytreated by methanolysis reaction so as to hydrolyze all the glycosidiclinks between sugars. Released monosaccharides were separated from thepeptide structure and sorted using a coupled Gas Chromatography-MassSpectrometry Automass apparatus (Finnigan). Molar ratio was determinedin reference to a known internal standard and to a 3 Mannose content ofa classical mammalian N-Glycan.

Such an analysis gave the following molar ratio for CHO-derived hIL-7

TABLE 5 Monosaccharide Fuc Gal Man GalNAc GlcNAc NeuAc Molecular Mass164 180 180 221 221 309 Peack Surface 43382 179120 310124 33650 344476423587 No. of nanomoles 5.41 24.76 22.15 5.26 27.29 20.94 Molar ratio0.73 3.35 3 0.71 3.69 2.83

Sites-specific N-glycan pattern heterogeneity of the CHO-derived hIL-7was assayed by endoprotease digestion, followed by fractionation andMass Spectrometry analyses of the generated peptides.

Purified samples were digested with Tripsin or other endo-proteases soas to generate glycopeptides corresponding to each N-glycosylation siteof the expressed IL-7. Each glycopeptide was identified by N-terminalmicrosequencing and by its specific retention time when analyzed byreverse phase HPLC. Each glycopeptide was therefore purified from theother ones. The heterogeneity of the N-glycans born by the glycopeptidewas analysed by MALDI-TOF MS (Q Star, Applied Biosystems). The m/zvalues corresponding to each peak of the MS spectrum allowedidentification of the N-Glycan pattern at a designated site of thehIL-7.

O-glycosylation was assayed via the use of O-glycan specific lectins(Lectin Blot, see FIG. 11).

Purified CHO-derived hIL-7 samples were separated by SDS-PAGE analysisand blotted to PVDF membranes. Immobilized proteins were probed with(but not limited to) peroxidase-labelled PNA (peanut agglutinin) and/orMAA (Maackia amurensis agglutinin) and stained for visualization.

Glycan heterogeneity and composition were also determined via the use ofLectin affinity to the purified CHO-derived hIL-7.

An array of lectins having affinity for N- and O-glycan structures wasselected and used to coat 96 well microplates. Identical amounts ofrecombinant purified IL-7 preparations were incubated into lectin coatedmicroplate wells. During this step, according to the affinity of a givenlectin to the glycan decoration of IL-7, different amount of IL-7 werekept bound to the lectin. Revelation was conducted by incubating an IL-7specific antibody coupled to Biotin. The Lectin-IL-7-Ab sandwich wasrevealed with a streptavidin-peroxidase conjugate.

Eight different lectins were used to characterize the IL-7 purifiedsamples. Each lectin specifically recognizes sugar moieties. Glycanmotifs and structure specificity are presented in Table 6.

TABLE 6 Table 6: summary of the pattern of sugar moieties recognized bylectins and inventory of their glycan motifs and structure specificity.Neu Gal Name Glc GlcNAc Man Fuc Ac NAc Gal Glycans structure specificityLEA + GlcNAcβ4GlcNAc and N- acetyllactosamine oligomers WGA + + GlcNAc,core of N-linked Glycans, Neu5ac UEAI + Fucose MAA +Neu5Acα-3Galb4GlcNAc- ACA + Galb3GalNAcα-O—R (T-antigen) AIA + Gala6 orGalβ3GalNAc (T-antigen), lactose ABA + Gal-GalNAcα-O—R, O-linked glycansPHAL Galb4GlcNAcβ6Man, branched complex N-glycans LEA is the lectin fromLycopersicon esculentum, WGA from Triticum vulgare, UEA.I from Ulexeuropeus, MAA from Maackia amurensis, ACA from Amaranthus caudatus, AIAfrom Artocarpus intergrifolia, ABA from Agaricus bisporus, PHA.L fromPhaseolus vulgaris.Results are presented in FIG. 12.

Lectins clearly demonstrate differential affinity, providing informationon the general structure of the accessible glycan decoration of thepurified IL-7 protein in solution.

Thus, ACA, ABA and AIA have affinity for Gal and GalNAc. All threelectins respond positively suggesting the presence of N- and O-Glycanstructures bearing these monosaccharides. The specific signal obtainedwith ABA reveals the presence of O-glycans structures. ACA has a weaksignal compared to AIA and to a lesser extent ABA. This reveals that the0 glycans are extended with little GalNAc as terminal residue.

LEA has affinity for GalNAc indicating the presence of N-Glycanstructures. Among the GlcNAc-specific lectins tested (data not shown),only those with affinity for N-acetyllactosamine reveal a positivesignal.

WGA presented a weak signal due to a low binding affinity to corestructures of N-linked glycans. Highly complex N-Glycans mask the corestructure and render lectin affinity difficult to operate.

UEA.I has specific activity to the presence of branched fucose. Bindingis rather weak indicating an uncomplete but effective fucosylation ofthe N-Glycans.

MAA has affinity to terminal sialic acids. MAA signal is strongindicating an effective sialylation on both N and O-glycans.

PHA.L has affinity to complex branched structures of N-Glycan and showeda strong signal, corroborating the results of MAA. PHA-L signal suggeststhe presence of large tri or tetra-antennary N-glycans.

Most typical mammalian O-glycans characterized on CHO-derived hIL-7(when applicable):

Altogether, these analyses indicate that the CHO cell-based expressionsystem used generates human IL-7 complex (triantennary) N-linkedoligosaccharide, as depicted in the following figure, branched to theirASN residue at position 70, 91 and 116 with high partial to completesialilation, up to 10 sialic acid residues. Also, the CHO-derived IL-7contains an O-glycan at position T110.

Therefore, although bearing complex sialilated N-glycans and O-glycans,the IL-7 purified batch still contains a mixture of fully and partiallyglycosylated proteins.

Example F Drug Substance to Drug Product: Formulation, Storage and LongTerm Stability of the Recombinant CHO Cell Expressed HIL-7

Search for optimal formulation of the drug substance was conductedthroughout a combinatory matrix study to evaluate the impact of variousstress conditions (temperature, buffer, pH, tonicity modifierconcentration, agitation, intense illumination) on the long termstability of the purified protein.

Highly complex purified recombinant human IL-7 was shown to be stable inAcetate as well as succinate buffers, at a concentration ranging between5 to 50 mM. Adequate pHs were chosen from pH=5.0 to 7.0 and idealstorage temperatures were between −20° C. to +4° C.

Sugars and low concentration of surfactants (Polysorbate polymers) maybe added to the preparation to prevent non covalent soluble aggregation.

In such conditions, IL-7 could be stored at +4° C. (in liquid form) at aconcentration ranging from 0.5 to 8.0 mg/ml, preferably from 2.0 to 4.0mg/ml, for more than 12 months. The pharmaceutical composition in liquidform has an improved stability profile.

Example G Proliferative Activity Analysis of Mammalian Cells-DerivedRecombinant Human IL-7 in a Specific Bioassay

The biological activity of mammalian cell-derived recombinant human IL-7was evaluated in a specific bioassay onto a murine pre-B cell linederived from bone marrow cells from CBA/C57BL mice, PB-1 (German CellBank DSMZ, Deutsche Sammlung von Mikrooganismen and Zellkulturen),strictly dependent on IL-7 for growth (Mire-Sluis et al.; 2000; J.Immunol. Methods; 236:71-76). These cells were maintained in culture incommercial IL-7 containing medium and starved for IL-7 prior toconducting the bioassay.

Bioassays were run with IL-7 samples to be tested, in parallel to aknown E. coli-derived IL-7 positive control and a negative controllacking IL-7.

IL-7, from control or samples, added to the starved cell culture,induced the dose dependent re-initiation of cell proliferation duringwhich radiolabelled thymidine (3H-Tdr, Amersham) was incorporated bydividing cells. The amount of labelling was pulsed and measured incounts per minute (cpm) in a liquid scintillation Beta counter(Wallack).

Alternatively, this bioassay may be conducted while using a dye markerreflecting the general metabolism of the cell, such as MTT dye(3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium, reduced bymitochondrial RedOx activity) or MTS dye(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium).

Serial dilutions of both the positive control and the samples to betested allowed plotting the number of cpm in relation to the amount ofsample/control assayed.

FIG. 13 presents dose-response kinetic data and curves obtainedroutinely in a typical bioassay: the PB-1 cell growth was induced byunglycosylated r-hIL-7 (expressed in E. coli) or highly glycosylatedr-hIL-7 (produced in mammalian cells). (Data points represent themean±SD of triplicate determination).

FIG. 14 presents dose-response kinetic data and curves obtainedroutinely in a typical bioassay: the PB-1 cell growth was induced byunglycosylated r-hIL-7 (expressed in E. coli), highly glycosylated orhyperglycosylated r-hIL-7 (produced in mammalian cells). (Data pointsrepresent the mean±SD of triplicate determination).

The important parameter to be considered for each sample was theED50=concentration (ng/ml) giving half-maximal activity. A higher ED50meaning a lower activity.

Activity comparability between IL-7 batches is addressed via theanalysis of the dose response curve parameter, such as slopecoefficient, maximal activity. From all curve parameters an ED50concentration (in ng/ml) pools parameters variation together. ED50corresponds to the IL-7 dose necessary to induce one half of thepossible maximal induction activity in vitro. In this regard, highlybioactive molecules correspond to low ED50 values whereas higher ED50concentrations will be typical from less bioactive IL-7 preparations invitro.

Nevertheless, in vitro bioactivity differences are not necessarilyrepresentative of similar in vivo bioactivity differences in the presentinvention.

Example H In Vivo Evaluation the Immunogenicity of HyperglycosylatedIL-7 Polypeptide in Primates

Simian hyperglycosylated IL-7 (sIL-7) expressed in CHO cell line(Examples A2, A6 and B) and purified according to Example D, wasevaluated in vivo for occurrence of potential immunogenicity, followingsIL-7 repeated administrations in normal primates.

Naïve young adult Cynomolgus monkeys (Macaca fascicularis) (n=4) wereentered into the study and received hyperglycosylated sIL-7 at the doselevel of 100 μg/kg/injection. Treated animals received a total of 6subcutaneous administrations of IL-7 over a period of five consecutiveweeks. The animals were clinically observed over a two month period.Blood specimens were collected, at different time points, throughout thestudy: on day 1 before sIL-7 administration, on day 37 and at the end ofthe study.

All animals survived the study and had no adverse reaction to sIL-7therapy. Administration of sIL-7 was locally well tolerated. When testedby interference in a specific ELISA assay aimed at detecting bindingantibodies, no anti-IL-7 antibodies were detected in the serum of alltreated animals. In comparison, E. coli-derived recombinant IL-7,although produced as a highly purified drug product, induced, in similarprotocol, the production of high titers of IL-7-binding antibodies insera ranging from 1:400 up to 1:5000.

Example I In Vivo Biological Activity of Hyperglycosylated IL-7Polypeptide in Primates

Human hyperglycosylated IL-7 (hIL-7) expressed in CHO cell line(Examples A1, A6 and B) and purified as in Example D, was evaluated invivo for determination of pharmacokinetic and pharmacodynamic profilesof hIL-7 in normal primates.

Naïve young adult Cynomolgus monkeys (Macaca fascicularis) were enteredinto the study and divided into two groups: untreated n=2 and hIL-7 100μg/kg/injection n=2. The treated animals received single subcutaneousadministration of hIL-7. The animals were clinically observed during 45days. Blood specimens were collected, at different time points,throughout the study: on days 1 (0, 3, 6, 9 and 12 hours postinjection), 2, 3, 4, 7, 21 and 45.

Administration of hIL-7 was well tolerated with no local reaction atinjection site. Following single subcutaneous administration of hIL-7 inmacaques, the pharmacokinetic pattern and parameters of hIL-7 wereestablished from the first 72 hours:

-   -   The plasma profile showed a bi-exponential decline after peak        absorption.    -   The observed product half-life in plasma was in the range of        30/40 hours. This half-life is significantly increased when        compared to the half-life observed with the E. coli-derived        recombinant IL-7 (5 to 8 hours) administered in the same        conditions. This reflects an improved in vivo stability of the        hyperglycosylated IL-7 polypeptide in blood.    -   The mean residence time (MRT) was 40 hours versus around 10 hrs        with the E. coli product.    -   The time to reach a maximum concentration was 180 minutes.

In conclusion, the pharmacokinetic study shows that thehyperglycosylated IL-7 polypeptide of this invention displays animproved and prolonged pharmacokinetic profile, which translates intoimproved pharmacodynamic effects.

The single injection of hIL-7 at 100 μg/kg induced a significantincrease in peripheral CD3⁺CD4⁺ and CD3⁺CD8′ T cell numbers,respectively 200% and 170% of changes from the baseline pre-treatmentvalues. The number of lymphocyte T cells (CD4 and CD8) expressing thespecific IL-7 receptor alpha chain (CD127) transiently decreases inperipheral blood as early as 6 hours post injection. Lymphocyte T cellsexpressing CD127 reappeared, in peripheral blood, 48 hours postinjection and returned to baseline values only 7 days post injection.Following single subcutaneous administration of E. coli-derivedrecombinant IL-7, the full return to baseline values of lymphocyte Tcells expressing CD127 occurred 4 days post injection. The kinetic ofreceptor occupancy of hyperglycosylated IL-7 polypeptide in more longercompared to E. coli-derived recombinant IL-7, reflecting the longerhalf-life of hyperglycosylated IL-7 polypeptide in primates as shownbelow. These results are in line with previous results showing thatalthough IV administration of IL-7 results in a better bioavailability,this does not translate into improved pharmacokinetic effects, in factthe extended delivery profile obtained by sub cutaneous injection ismore efficient than the acute delivery profile obtained after IVinjection. Here the hyperglycosylation of the protein induces aprolonged kinetic profile, which in turn translates into an improvedpharmacodynamic activity. In view of this extended profile also it isalso expected an improved clinical tolerance, because drug sides effectsare usually linked to peak concentrations.

1. (canceled)
 2. A hyperglycosylated IL-7 composition, wherein saidcomposition comprises a mammalian IL-7 polypeptide having at least threeglycosylated amino acid residues, an average isoelectric point less than6.5, an average molecular weight greater than 27 KDa as determined bySDS gel electrophoresis and the following three disulfide bridges: Cys:1-4 (Cys2-Cys92); 2-5 (Cys34-Cys129); 3-6 (Cys47-Cys141) and apharmaceutically acceptable carrier, excipients or diluents.
 3. Thecomposition of claim 2, wherein the glycosylated amino acid residues arelocated at glycosylation sites which are naturally present and/orartificially created in the IL-7 polypeptide sequence.
 4. Thecomposition of claim 3, wherein the glycosylation sites are selectedfrom Asn residues at positions 70, 91 and 116; Thr at position 110, anyartificially created glycosylation sites as listed in Table 1, or anycombination thereof.
 5. The composition of claim 2, wherein said IL-7polypeptide comprises N-linked carbohydrate selected from: a) amammalian type sugar chain; b) a sugar chain comprising a complexN-carbohydrate chain; c) a sugar chain sialylated byalpha2,6-sialyltransferase or alpha2,3-sialyltransferase; and/or d) asialylated sugar chain displaying between 3 to 30sialyl-N-acetylgalactosamine.
 6. The composition of claim 2, whereinsaid IL-7 polypeptide comprises O-linked carbohydrate chain(s) with aterminal sialic acid residue.
 7. The composition of claim 3, whereinsaid glycosylation sites are glycosylated with carbohydrate chains thatcomprise tetra-antenary to biantenary structures with partial orcomplete terminal sialylation.
 8. The composition of claim 7, whereinsaid glycosylation sites are glycosylated with carbohydrate chains thathave a tri-antenary structure and tri- or bi-sialylation and/or adiantenary structure with disialylation.
 9. The composition of claim 2,wherein said IL-7 polypeptide has an in vivo extended half-life and meanresidence time as compared to non-glycosylated IL-7 polypeptides. 10.The composition of claim 2, wherein said IL-7 polypeptide comprises oneor more of the following amino acid substitutions:Lys28Asn-Ile30Ser-Ile30Thr-Ile30Asn-Ser32Thr-Leu35Ser-Leu35Thr-Glu38Ser-Glu38Thr-Phe39Ser-Phe39Thr-Phe42Ser-Phe42Thr-Glu52Ser-Glu52Thr-Val82Asn-Glu84Thr-Glu84Ser-Lys97Asn-Arg99Thr-Arg99Ser-Ala102Asn-Leu104Thr-Leu104Ser-Leu104Asn-Glu106ThrGlu106Ser-Leu128Ser-Leu128Thr-Ile145Asn-Met147Thr-Met147Ser.
 11. Thecomposition of claim 2, wherein said mammalian IL-7 polypeptide is ahuman IL-7 polypeptide comprising of SEQ ID NO:
 1. 12. The compositionof claim 2, produced by: a) culturing, in a fed-batch or perfusion mode,a recombinant host cell comprising a recombinant nucleic acid moleculeencoding an IL-7 polypeptide; b) collecting the IL-7 polypeptideproduced from said cell; and c) purifying said IL-7 polypeptide by amethod comprising at least a step of hydrophobic interactionchromatography, ion exchange chromatography, affinity chromatography orgel filtration chromatography, either alone or in various combinations,so as to obtain a composition which comprises at least 80% mammalianIL-7 polypeptides having at least three glycosylated amino acidresidues, an average isoelectric point less than 6.5 and an averagemolecular weight greater than 27 KDa as determined by SDS gelelectrophoresis; and comprising the following three disulfide bridges:Cys: 1-4 (Cys2-Cys92); 2-5 (Cys34-Cys129); 3-6 (Cys47-Cys141).
 13. Ahyperglycosylated, IL-7 composition, wherein said composition containsmammalian IL-7 polypeptides having an average isoelectric point lessthan 6.5 and an average molecular weight greater than 27 KDa asdetermined by SDS gel electrophoresis, wherein said mammalian IL-7polypeptides comprise SEQ. ID NO: 1 with the following amino acidchanges: Lys28Asn and Ile30Ser; Lys28Asn and Ile30Thr; Ile30Asn andSer32Thr; Leu35Ser; Leu35Thr; Glu38Ser; Glu38Thr; Phe39Ser; Phe39Thr;Phe42Ser; Phe42Thr; Glu52Ser; Glu52Thr; Val82Asn and Glu84Ser; Val82Asnand Glu84Thr; Lys97Asn and Arg99Ser; Lys97Asn and Arg99Thr; Ala102Asnand Leu104Ser; Ala102Asn; Leu104Thr; Leu104Asn and Glu106Ser; Leu104Asnand Glu106Thr; Leu128Ser; Leu128Thr; Ile145Asn and Met147Ser; Ile145Asnand Met147Thr; or Met147Asn and Thr149Ser, and wherein said mammalianIL-7 polypeptides comprise the following three disulfide bridges: Cys:1-4 (Cys2-Cys92); 2-5 (Cys34-Cys129); 3-6 (Cys47-Cys141).